Nanopore for Educators eBook

A community-developed guide to Nanopore sequencing in the classroom

Welcome

This eBook is a community-authored guide to integrating Oxford Nanopore DNA sequencing into hands-on educational experiences. For around $2000 in startup costs, students can start sequencing DNA using the same tools as cutting-edge researchers.

Nanopore Quick Start

Learn why Nanopore sequencing is the most classroom-friendly platform for research-grade DNA sequencing and course-based research experiences.

Getting started
Save time and money

Curated equipment and reagent list, cost estimates, and planning guides.

Time and Cost Planning
Highly-annotated protocols

Educator-friendly protocols combine tips and detailed explanations.

16S Shoe-ome protocol
Questions answered

Join our community! Get advice from an expert or an AI.

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Why a guide for educators?

This guide is an educator-focused introduction to Nanopore DNA sequencing. Its primary goal is to share resources and approaches that make it easier to use Nanopore technology in the classroom, especially (but not exclusively) for course-based undergraduate research experiences (CUREs). Importantly, we aim to ensure that low-cost, easy-to-use DNA sequencing is accessible to institutions with fewer resources and to communities that haven't had full access to Science, Technology, Engineering, and Math (STEM) participation.

As a guide for educators, the content of this site is curated to focus on experiments and techniques well-suited for the classroom. We emphasize approaches that minimize costs and complexity, rather than those requiring the most well-equipped labs. Additionally, we aim to feature adaptable teaching resources (e.g., slides, lessons, protocols, videos, advice) to give educators a head start in developing their own teaching materials. The site is versioned to provide educators with a stable resource to rely on, even as updates are made.

What this guide is not

There are a few things to keep in mind when making use of this guide:

This guide is not intended to replace or supersede official protocols or warranty agreements.
It is not a publication of Oxford Nanopore Technologies. All content is contributed and curated by community members. While protocols and recommendations are tested, users assume all risks. We include, link to, and cite resources, including peer-reviewed articles for further exploration.
The guide is not a comprehensive exploration of all the capabilities of the Nanopore sequencing. While we include experiments and techniques that can be successfully used by a wide range of educators, we do not cover every potential application.

Nanopore Network

We are a community you can join!

Connect with other like-minded educators interested in using Nanopore in the classroom. Join our Slack channel for realtime discussion and connect with us through QUBES, where we organize semester-long "faculty mentoring networks." These are regular meetings of faculty members who share knowledge and experience about integrating Nanopore into teaching, with a focus on CUREs.

Join the Slack Join on QUBES

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Ask a question

Our experimental AI chatbot is trained on a variety of Nanopore reference materials, and peer-reviewed publications on Nanopore sequencing and undergraduate education. Ask a question to receive a synthesized answer, complete with references to the original sources. Please verify your understanding with a trusted source, as no AI bot can guarantee 100% accuracy.

Citation and licensing

Citation

Please use the following citation:

Jason J. Williams¹, Anna Alicja Feitzinger¹, Miguel A. Urdaneta-Colon², José L. Agosto-Rivera², James T. Melton III³, Jeremy Seto⁴, Carlos C. Goller⁵, Patrick Lypaczewski⁶, Jordan Dotson³, Luis E. Vázquez-Quiñones⁷, Ilana Ruth Cohen⁸, Trevor T. Duarte⁹, Katie M. Sandlin¹⁰, Maria B. Lazebnik¹¹, Mark A. J. Roberts¹², Jeffry Petracca¹, Yokshitha Bathula¹³, Dmitry Y. Brogun¹⁴, Jennifer Katcher¹⁵, Kaitlyn Parrish³, and Naupaka Zimmerman¹⁶. Nanopore for Educators eBook. Cold Spring Harbor (NY): Nanopore Network; Version April-2025. Available from: https://nanopore4edu.org; DOI: https://doi.org/10.5281/zenodo.15149313

Affiliations

¹ Cold Spring Harbor Laboratory, ² University of Puerto Rico, Río Piedras Campus, ³ Spelman College, ⁴ New York City College of Technology, City University of New York, ⁵ North Carolina State University, ⁶ McGill University, ⁷ Universidad Interamericana de Puerto Rico, Arecibo Campus, ⁸ City University of New York Graduate Center, ⁹ El Paso Community College, ¹⁰ Genomics Education Partnership, ¹¹ Bentley University, ¹² University of Oxford, ¹³ HudsonAlpha Institute for Biotechnology, ¹⁴ Kingsborough Community College, City University of New York, ¹⁵ Pima Community College, and ¹⁶ University of San Francisco.

Licensing: Unless otherwise noted, all information developed for an distributed through this site not separately attributed or under the terms of another license or usage agreement (e.g. publications and contributed resources) may be considered licensed under Creative Commons BY 4.0 Attribution 4.0 International. All information is presented for educational use and without warranty.

Funding acknowledgment, and declared interests

This material is based upon work supported by the National Science Foundation under Grant No. (DUE:2216349) as a Level-1 multi-institutional IUSE (Institutional and Community Transformation) project, we have assembled a diverse team of investigators to develop and test infrastructure to support genome science at three MSIs: New York City College of Technology, Spelman College, the University of Puerto Rico-Río Piedras. It is led by the Cold Spring Harbor Laboratory DNA Learning Center.

This site exclusively highlights the potential of Oxford Nanopore Sequencing because currently, it is the only DNA sequencing platform we believe provides an accessible and affordable hands-on platform for DNA sequencing by pre-college and college students. The authors declare no financial conflict of interest with Oxford Nanopore. At times, Oxford Nanopore has provided the authors and other educators with free or discounted materials exclusively for use in training and education as part of its educator discount program.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used OpenAI ChatGPT to generate summary content for various sections and for grammatical correction, proofreading, and formatting. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

CSHL logo CUNY City Tech logo University of Puerto Rico, Rio Piedras logo

NSF-Logo Funded by the U.S. National Science Foundation. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. DUE:2216349

Get Started ↵

Ten Steps to Getting Started with Nanopore

Essential steps for getting started with Nanopore sequencing

Understand the Basics of Nanopore Sequencing
- Familiarize yourself with how Nanopore sequencing works, its capabilities, and its limitations.
  - Review this book chapter: The Current State of Nanopore Sequencing 2023
  - View Nanopore's video: How to get started with nanopore sequencing and plan your experiment
  - Review advanced research Applications making use of Nanopore sequencing.
Assess Your Curriculum Goals
- Define the objectives for integrating Nanopore sequencing into your classroom.
- Determine whether you will focus on specific applications (e.g., microbial diversity, genome assembly, or barcoding) or introduce students to the general principles of sequencing.
- Know benefits and strategies for introducing course-based research experiences.
  - Find resources for developing undergraduate course-based research experiences: Fear of the CURE: A Beginner’s Guide to Overcoming Barriers in Creating a Course-Based Undergraduate Research Experience
Plan and Budget for Equipment and Materials
- Use our list of essential equipment (e.g., MinION sequencer, magnetic racks, pipettes) and reagents (e.g., DNA extraction kits, sequencing kits).
  - We recommend starting with the 16S demonstration experiment or DNA barcoding demonstration experiment to get started. Each experiment details all the needed materials.
  - Use educational discounts where available to minimize costs.
  - Check with colleagues at your institution or nearby who may be using Nanopore and can provide you with support.
Set Up Online Accounts and Software
- Register for an Oxford Nanopore Technologies account.
  - Orders for Nanopore devices, library kits, reagents and accessories at the Nanopore store.
    - You will likely need to coordinate with your purchasing department to setup shipping and payment.
    - Orders can also be placed through an authorized vendor such as Avantor; check with Nanopore on additional options.
  - You will be able to purchase a starter pack to get your device.
- Explore free platforms like Galaxy, DNA Subway, and CyVerse for bioinformatics workflows.
Acquire Hands-On Experience
- Conduct trial runs of Nanopore workflows before teaching.
- Practice key steps such as DNA extraction, library preparation, and sequencing.
- Use mock samples or pre-prepared datasets to familiarize yourself with bioinformatics tools.
Develop or Adapt Teaching Materials
- Prepare slides, handouts, and protocols tailored to your specific learning objectives.
- Include clear instructions for students on safety, experimental techniques, and data analysis.
- Incorporate assessments to evaluate students’ understanding and progress.
Engage in Protocol Optimization
- Test protocols with your lab aides or instructional staff to identify potential bottlenecks.
- Adjust protocols for compatibility with your class size, time constraints, and available resources.
Plan Your First Experiment
- Choose a simple and achievable experiment (e.g., sequencing microbial DNA or barcoding plant species).
- Use small-scale experiments to build student confidence and refine your approach for larger projects.
- Layout how sequencing work will be divided between individual students, student groups, and instruction staff.
Leverage Community Resources
- Join educator-focused communities like our QUBES network and Nanopore Network Slack.
- Attend faculty mentoring networks, webinars, or in-person training sessions to exchange knowledge and strategies with peers.
Evaluate and Iterate
- After your first experiments, evaluate student outcomes and overall workflow.
- Gather feedback from students and instructional staff to refine future experiments and lesson plans.
- Document your experience to build a robust and repeatable curriculum.

Estimating time and costs to get started with Nanopore

Integrating Nanopore sequencing into a classroom or teaching laboratory offers many benefits. However, estimating the time and total costs required to get started is essential for successful planning and implementation. This section provides some tips and observations to help educators understand the resources and time needed to bring Nanopore sequencing to their students.

When estimating costs, educators should consider both initial and recurring expenses. These include purchasing essential equipment, consumables, reagents, and sequencing kits, as well as ensuring access to appropriate computational resources. Time estimates should account for equipment setup, instructor preparation, and the time required to guide students through laboratory protocols and bioinformatics analyses.

It’s important to note that these are general guidelines, and several factors can influence both the time and cost of implementation. For example:

Class size and the number of sequencing projects planned may affect the quantity of consumables and reagents required.
Availability of existing laboratory equipment and computational resources can significantly reduce start-up costs.
Instructor experience with molecular biology techniques and bioinformatics may influence preparation time and the level of support needed.
Institutional purchasing agreements or discounts may reduce the costs of equipment and reagents.

Example prep and teaching activities and timings

Activities	Estimated Duration	Items/Activities to Add Up Costs($)	Notes
Background research on Nanopore sequencing	(1–2) days	Training workshops (free-$ for travel or tuition)	Time for familiarization; consider workshops or online resources
Curriculum planning	(1–2) weeks	Lesson plans, assessments, teaching materials	Aligns activities with course objectives; includes material development
Protocol testing	(1) month	Reagents ($$$), consumables ($$), staff time	Trial runs with aides or TAs; optimize protocols for classroom
Sample collection	(1) 2-hour lab section	Collection supplies ($), travel ($) and equipment ($$)	Depends on sample type (e.g., environmental, lab specimens)
DNA extraction	(1–2) 2-hour lab sections	Reagents ($$), consumables ($), equipment ($$$)	Includes DNA isolation and quality checks; depends on sample type
Library preparation	(1-2) 2-hour lab section	Sequencing kits ($$), consumables ($), reagents ($$)	Laboratory staff or instructor pools samples and generates libraries
DNA sequencing	(1) 2-hour lab section	Flow cells ($-$$), sequencing kits ($$), compute resources	Some samples may require 1-hour runs; others may need 24+ hours
Data analysis	(2) 2-hour lab sections	Software subscriptions ($), cloud resources (free-$)	Includes bioinformatics tools and interpretation; longer steps like basecalling occur outside class time
Evaluation and feedback	(1) week	Instructor time, survey tools, debriefing materials	Grading assessments, collecting feedback, and debriefing instructional staff

Example costs - Nanopore equipment

This table outlines the estimated minimal startup costs for purchasing your first set of Nanopore sequencing materials, assuming that you already have most basic molecular biology reagents and equipment. The potential education discounts are included for illustrative purposes only—contact Oxford Nanopore to inquire on available education discounts.

Item	Section/Class Quantity	Base Cost	Total at Base Cost	Education Discount Price*	Total w/Discount
MinION Startup Pack (MinION flow cell Flongle flow cells, sequencing kit)	1	$2,000	$2,000	$1,600	$1,600
Flongle and adapter	1	$1,460	$1,460	0 (included in startup pack)
Magnetic rack	6	$59	$354	$59	$354
MacBook computer	1	$2,800	$2,800	$2,400	$2,400
Total Startup Costs			$6,614		$4,354

*The provided educational discounts are illustrative and may vary depending on institutional agreements or vendor promotions

Example costs - Nanopore reagents and consumables

This table provides an overview of the estimated costs for different Nanopore sequencing kit types and associated reagents when used in classroom settings. It includes base costs, costs per sample or reaction, and potential educational discounts. The table highlights the potential costs for a class of 24 students, assuming standard consumables and reagents.

16S Kit (6 x 24 samples) - Microbial diversity

Nanopore Sequencing Kit Type	Base Cost	Total per Sample/Run	Education Discount Price*	Total per Sample/Run w/Discount
Nanopore 16S Kit	$900.00	$6.25	$400.00	$2.78
Norgen DNA extraction kit and swabs (50 preps)	$638.00	$12.76	$638.00	$12.76
NEB LongAmp Taq (100 rxn)	$161.00	$1.61	$128.00	$1.28
Flongle (12 pack - 1 Flongle per run)	$67.00	$2.79	$67.00	$2.79
Consumables ($4 per student)	$96.00	$4.00	$96.00	$4.00
Potential cost per student/run		$27.41		$23.61
Total for class of 24		$657.88		$566.63

Rapid Barcoding Kit (6 x 24 samples) - DNA barcoding and small genomes

Nanopore Sequencing Kit Type	Base Cost	Total per Sample/Run	Education Discount Price*	Total per Sample/Run w/Discount
Nanopore Rapid Barcoding Kit	$699.00	$4.85	$110.00	$0.76
Low-cost "homemade" DNA extraction (24 preps)	$24.00	$1.00	$24.00	$1.00
Low-cost Taq and PCR reagents (24 preps)	$50.00	$2.08	$50.00	$2.08
Flongle (12 pack - 1 Flongle per run)	$67.00	$2.79	$67.00	$2.79
Consumables ($4 per student)	$96.00	$4.00	$96.00	$4.00
Potential cost per student/run		$14.73		$10.64
Total for class of 24		$353.50		$255.33

Ligation Kit (6 samples) - Small genomes

Nanopore Sequencing Kit Type	Base Cost	Total per Sample/Run	Education Discount Price*	Total per Sample/Run w/Discount
Nanopore Ligation Kit	$699.00	$233.00	$110.00	$36.67
DNA extraction kit (example, NEB Monarch) 50 samples; 6 runs	$517.00	$20.68	$413.60	$16.54
NEB companion module (24 reactions); 6 runs	$1,025.00	$85.42	$820.00	$68.33
MinION flow cell (1 per run - assuming 2 runs, washed and reused)	$750.00	$375.00	$450.00	$225.00
Consumables ($4 per student)	$96.00	$4.00	$96.00	$4.00
Potential cost per student/run (2 runs or non-barcoded samples, 24 students)		$718.10		$350.54
Total for class of 24		$718.10		$350.54

*The provided educational discounts are illustrative and may vary depending on institutional agreements or vendor promotions

Comments and discussion

See recent comments or start a discussion on our Slack channel.

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Start-up Checklists

To run a successful Nanopore sequencing experiment in your classroom, you’ll need a combination of basic molecular biology tools, a Nanopore sequencing device, and computational resources. Below is an overview of the materials required, with notes on equipment we generally consider essential. Some specific recommendations also include links to products we have had good success with.

Warning

Check your protocol

These lists are generalizations. Review the specific protocols for your experiments to verify you have the needed supplies. Also note, that PPE recommendations are also specific to the materials and reagents you will use, as well as you institution's laboratory safety guidelines. Please follow those specific guidelines.

Equipment, reagents, supplies, and computer technologies lists

Personal Protective Equipment

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
PPE	Disposable gloves	Yes	In addition to reagent contact, recommended to avoid cross contamination	1 pair per student, lab aide, and instructor per session	Nitrile gloves	$10–$30 per box (100)
PPE	Lab coats		Ensures personal and sample safety	1 per student, lab aide, and instructor	Standard lab coats	$20–$50 per coat
PPE	Safety goggles		May be called for during sample collection or DNA extraction.	1 per student, lab aide, and instructor	Chemical splash goggles	$5–$20 per goggle
PPE	Waste disposal bags		For safe disposal of biological waste	Varies	Biohazard disposal bags, especially needed if working with cultured microbial samples	$10–$30 per pack

Molecular Biology Equipment

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Equipment	Micropipettes (1-10 µl, 10-100 µl, 100-1000 µl)	Yes	For precise liquid handling in Nanopore protocols.	1 set per group + 1 for instructor	Adjustable-volume pipettes from reliable brands like Eppendorf or Gilson.	$50–$300 per pipette
Equipment	Thermocycler	Likely	Supports 24+ barcoded samples. Gradient PCR capability is recommended for optimization.	1 per lab	Bio-Rad or Thermo Fisher systems are suitable for classroom use. Smaller units by miniPCR are inexpensive.	$700–$7,000
Equipment	Microcentrifuge	Yes	For 12-24 1.5 ml tubes. Smaller format for PCR tubes is helpful but not essential.	1 per lab	Tabletop models from Eppendorf or Thermo Fisher.	$200–$2000
Equipment	Water bath or heat block	Yes	For incubation steps. Hotplates may be used for preparing electrophoresis gels.	1 per lab (some protocols benefit from additional units)	Heat blocks with adjustable temperature settings are versatile for multiple protocols.	$150–$800
Equipment	Microwave		Hotplates may also be used for preparing electrophoresis gels.	1 per lab	Standard lab microwave or hotplate.	$50–$150
Equipment	Electrophoresis tank and power source	Likely	Recommended for checking PCR products and, in some cases, DNA quantity and quality.	1 set per group or 1 or more communal sets	Compact electrophoresis systems are recommended. See sets such as Carolina and MiniPCR QP-1600-01.	$200–$1000
Equipment	Electrophoresis documentation setup	Likely	UV light source for gel visualization; camera setup (can include a cell phone with proper filters).	1 per lab	Blue light transilluminators are a safe alternative to UV setups.	$300–$800
Equipment	Glassware for agarose gels (100 ml cylinder, 200 ml Erlenmeyer flask)	Likely	Required for gel preparation.	1 set per lab	Durable glassware from lab supply companies.	$20–$50 per item
Equipment	Magnetic rack	Yes	For magnetic bead cleanups. Should support 1.5–2.0 ml tubes.	1 per group and 1 or more for lab aid and instructor	Sergilabs offers affordable options. Amazon link	$30–$100
Equipment	Ice bucket(s)/styrofoam containers	Yes	For keeping reagents and samples cold during preparation.	1 per group and several for lab use	Reusable or disposable options depending on budget.	$10–$50
Equipment	-20°C non-defrosting freezer	Yes	Essential for storing reagents and DNA samples. Low-cost domestic units can be substituted ($200-300) must be non-defrosting.	1 per lab	Lab-grade non-defrosting freezers are essential to prevent degradation of sensitive materials.	$1,000–$3,000
Equipment	4°C fridge	Yes	For short-term reagent and sample storage as well as storage of flow cells. Low-cost domestic units can be substituted ($200-300).	1 per lab	Lab-grade refrigerators preferred for consistency.	$1,000–$2,000
Equipment	Qubit fluorometer		For DNA quantification. Many protocols will work with estimates provided by electrophoresis gel.	1 per lab (optional)	Thermo Fisher Qubit (Q33238). Thermo Fisher link	~$4,600
Equipment	Nanodrop		For DNA quantification. Similar considerations as the Qubit.	1 per lab (optional)	Thermo Fisher Nanodrop (NDLPLUSGL). Thermo Fisher link	~$7,200

Nanopore Sequencing Equipment

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Sequencing Equipment	MinION sequencer (M1kC or M1kD)	Yes	Portable device capable of generating up to 50 Gb per run. Note: M1kB model is discontinued.	1 per group or lab	Suitable for a wide range of applications; choose model based on specific needs.	~$1,000–$2,000*+
Sequencing Equipment	Flongle adapter	Likely	Adapter for MinION or GridION; enables sequencing on Flongle Flow Cells, generating up to 2.8 Gb per run.	1 per group or lab	Ideal for small-scale, cost-effective sequencing projects.	~$1,460* - generally available only as part of a starter pack
Sequencing Equipment	GridION		High-throughput device; can run up to 5 MinION Flow Cells simultaneously, each generating up to 50 Gb per run.	1 per lab (if applicable)	Suitable for labs requiring higher throughput and integrated compute capabilities.	~$67,000*+
Sequencing Equipment	P2 Solo		Modular device; can run up to 2 PromethION Flow Cells simultaneously, each capable of generating up to 290 Gb per flow cell. Note: P2 and PromethION flow cells are different and not compatible with MinION flowcells.	1 per lab (if applicable)	Ideal for labs with high-throughput sequencing needs; requires external compute resources.	~$9,555*+

Tip

Educator Discounts*

Prices change frequently, and these prices do not necessarily reflect potential educator discounts as they are offered by Oxford Nanopore.

Nanopore Sequencing Kits

Depending on your application, you will need at least one Nanopore library preparation kit. We feature the following in this guide, see the Nanopore Store for a full product list.

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Sequencing Kits	Ligation Sequencing Kit V14 (SQK-LSK114)	Yes	Flexible method for preparing sequencing libraries from dsDNA, including gDNA, cDNA, or amplicons. Involves end-repair, dA-tailing, and adapter ligation.	1 per run (6 genomic samples)	Whole-genome sequencing, amplicon sequencing, and cDNA sequencing.	$699*
Sequencing Kits	Rapid Barcoding Sequencing Kit 24 V14 (SQK-RBK114.24)	Yes	Enables rapid preparation of up to 24 barcoded sequencing libraries without PCR amplification. Suitable for low-input gDNA.	1 per run (24 barcoded samples)	DNA barcoding, microbial genomics, small genome sequencing, and amplicon sequencing.	$699*
Sequencing Kits	16S Barcoding Kit 24 V14 (SQK-16S114.24)	Yes	Designed for rapid amplification and sequencing of the 16S rRNA gene, allowing genus-level identification of bacteria. Includes 24 unique barcodes for multiplexing.	1 per run (24 barcoded samples)	Microbiome analysis and bacterial identification.	$900*

Tip

*Educator Discounts

Prices change frequently, and these prices do not necessarily reflect potential educator discounts as they are offered by Oxford Nanopore.

Tip

Nanopore Library Prep Kits

We will provide specific recommendations for Nanopore sequencing kits which have their own specific reagents and associated costs. Additional reagents required by the kit incur additional costs.

Molecular Biology Consumables

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Consumables	Micropipette tips (1-10 µl, 10-100 µl, 100-1000 µl)	Yes	Consider filter tips for PCR and/or with precious samples to avoid cross-contamination.	Multiple boxes per lab	Filter tips recommended for sensitive work.	$10–$50 per box
Consumables	0.2 ml PCR tubes	Yes	Essential for PCR workflows.	Multiple packs per lab	High-quality, sterile PCR tubes from trusted brands.	$10–$30 per pack
Consumables	1.5 ml or 1.7 ml microfuge tubes	Yes	Sterile and DNA/DNase-free strongly recommended.	Multiple packs per lab	Brands like Eppendorf or Thermo Fisher offer reliable options.	$10–$30 per pack
Consumables	1.7 ml lo-bind tubes		Many Nanopore protocols call for these. Strongly recommended when working with precious samples.	Multiple packs per lab	Eppendorf LoBind Tubes	~$50 per box (250)
Consumables	Freezer-safe tape and labels	Yes	For labeling samples stored at -20°C or lower.	Multiple rolls per lab	Freezer-safe tape and lab-specific labels ensure sample integrity.	$5–$20 per roll
Consumables	Permanent markers	Yes	Useful for labeling tubes and containers.	Multiple per lab	Choose markers with fade-resistant, waterproof ink.	$1–$5 per marker

Molecular Biology Reagents

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Reagents	Agarose powder	Likely	For preparing electrophoresis gels.	Multiple packs per lab	Molecular biology grade agarose is recommended.	$50+ per bottle
Reagents	DNA loading dye	Likely	Required for visualizing DNA on electrophoresis gels.	1–2 bottles per lab	Choose dyes compatible with your gel documentation system.	Varies, but inexpensive
Reagents	DNA stain	Likely	For staining DNA in gels. Options include SYBR Safe, GelRed, or ethidium bromide (use cautiously).	1–2 bottles per lab	Invitrogen SYBR Safe is non-toxic; GelRed is safer than ethidium bromide and has good visualization properties.	$100–$300 per bottle
Reagents	Buffer solutions (e.g., TAE or TBE)	Likely	For electrophoresis and sample preparation.	Multiple bottles per lab	Purchase premade buffers or prepare stock solutions.	$20–$50 per bottle
Reagents	DNA standards for electrophoresis	Yes	For estimating DNA fragment sizes during gel electrophoresis.	1–2 vials per lab	Use molecular weight ladders appropriate for your protocol, such as 1 kb or 100 bp ladders.	$20–$100 per vial
Reagents	Nuclease-free water	Yes	For preparing samples and dilutions.	Multiple bottles per lab	Molecular biology grade water is recommended for all DNA-related protocols.	$50+ depending on quantity
Reagents	200 proof ethanol	Yes	Used in DNA/RNA purification and sample cleanup protocols.	Multiple bottles per lab	Molecular biology grade ethanol ensures no contamination in sensitive workflows.	$50+ depending on quantity
Reagents	Isopropanol	Yes	Commonly used in DNA/RNA precipitation protocols.	Multiple bottles per lab	Molecular biology grade isopropanol is preferred for reliable results.	$50+ depending on quantity

Tip

DNA Extraction Kits

We will provide specific recommendations for DNA extraction kits and protocols which have their own specific reagents and associated costs.

Computers, accessories, software, and online accounts

Category	Item	Essential	Notes	Quantities	Recommendations	Price Range
Computers and Accessories	Laptop or desktop computer*	Yes	Required for running sequencing software and data analysis tools.	1 per lab and/or per sequencing device	Mid-range or higher specifications; 16 GB RAM, quad-core processor recommended.	$1,000–$2,800
Computers and Accessories	External storage		Useful for storing large sequencing data files.	Several hundred GB to 1TB+ depending on use	Portable SSDs with fast read/write speeds are ideal.	$100–$500
Computers and Accessories	Internet connection	Yes	Required for downloading software updates and accessing cloud-based tools.	1 computer	Stable broadband is sufficient; Wi-Fi or Ethernet compatible.	Varies
Software	MinKNOW	Yes	Primary software for operating Oxford Nanopore sequencing devices.	1 per computer	Comes with Oxford Nanopore devices; ensure latest version is installed.	Free with devices
Software	EPI2ME	Likely	Cloud-based data analysis platform for processing sequencing data.	1 installation per computer	Useful for beginners or labs without bioinformatics expertise.	Free or subscription
Online Accounts	Oxford Nanopore account	Yes	Required for purchasing devices, kits, and accessing resources.	1 per instructor	Sign up through the Oxford Nanopore website.	Free
Online Accounts	Cloud storage (e.g., Google Drive, Dropbox)		Useful for sharing and backing up sequencing data. Must have an allocation manager who can share access to their resources.	1 per lab	Choose a storage platform with adequate space for large datasets.	Free or subscription
Online Accounts	NSF ACCESS-CI		National computing infrastructure for advanced data processing.	Depends on configuration	Apply through ACCESS-CI for computational resources.	Free
Online Accounts	JetStream2		Cloud computing resource for scientific workflows. Requires an NSF ACCESS-CI account and allocation for at least one account manager; students will not necessarily need their own account.	1 allocation per instructor (can be used across multiple students/sections)	Apply through JetStream2 for access to cloud-based computation.	Free
Online Accounts	DNA Subway		Educational platform for bioinformatics and genomics analysis.	1 account per student, instructor, lab aide	Sign up at DNA Subway for classroom-friendly analysis tools.	Free
Online Accounts	CyVerse		Cloud-based platform for managing and analyzing large biological datasets.	1 account per student, instructor, lab aide	Apply at CyVerse for access to computational and data management tools.	Free or subscription
Online Accounts	Galaxy		Open-source platform for bioinformatics workflows, accessible via a web interface.	1 account per student, instructor, lab aide	Perform bioinformatics tasks without the need for local installations using Galaxy.	Free

Tip

Choosing a Computer to Run a MinION Device*

Selecting the right computer for your MinION device is crucial for smooth sequencing and data analysis. The requirements vary slightly depending on the model you're using and the operating system:

For the MinION M1kB

Operating System: Windows 10 or 11, macOS Monterey (12), Ventura (13), Sonoma (14), or Ubuntu 20.04 or 22.04.
Processor: Intel or AMD processor with at least 4 cores/8 threads.
Memory: 8 GB RAM (minimum).
Storage: 500 GB SSD or higher for managing sequencing data.
USB Ports: Requires USB 3.0 port.

For the full IT requirements, refer to the MinION M1kB specifications.

For the MinION M1kD

Operating System: Windows 10 or 11, Ubuntu 20.04 LTS, or macOS 12 (Monterey) or later.
Processor: Intel or AMD processor with at least 4 cores/8 threads.
Memory: 16 GB RAM or more for optimal performance.
Storage: 1 TB SSD or higher to handle large datasets.
USB Ports: Requires USB-C port.

For the full IT requirements, refer to the MinION M1kD specifications.

General Recommendations

Portability: A laptop or MacBook is ideal for portability, especially for fieldwork or classroom use.
Performance: For labs handling high-throughput sequencing, invest in a computer with higher specifications than the minimum.
External Storage: Consider adding an external SSD to manage and back up sequencing data efficiently.

Choosing the right system ensures reliable performance and smooth operation of your MinION device.

Current best pick - MacBook Pro

While any computer meeting the minimum specifications provided by Oxford Nanopore Technologies will work, we currently recommend the 14-inch MacBook Pro with the M3 chip for its excellent performance and compatibility. This recommendation may be updated as technologies evolve. If you already have access to a computer meeting Nanopore's minimum requirements, it should work just fine.

Recommended Configuration

Processor: Apple M3 chip with 8-core CPU, 10-core GPU, and 16-core Neural Engine.
Memory: 24 GB unified memory.
Storage: 1 TB SSD for managing sequencing data.
Ports: Two Thunderbolt / USB 4 ports, HDMI port, SDXC card slot, headphone jack, and MagSafe 3 port.
Operating System: macOS Sonoma (14) or later.
Display: 14-inch Liquid Retina XDR for clear and detailed data visualization.

Learn more about this MacBook Pro model on the Apple website.

Why We Recommend This MacBook Pro

Performance: The M3 chip provides robust performance for running sequencing software like MinKNOW and performing bioinformatics analyses.
Portability: Its compact size and long battery life make it ideal for classroom or fieldwork settings.
Compatibility: Fully compatible with Oxford Nanopore devices and software, including macOS-supported versions.
Future-Proofing: With cutting-edge technology, this MacBook is well-suited for evolving software and sequencing demands.

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Ended: Get Started

Equipment and Lab Management ↵

Oxford Nanopore Sequencing Hardware

The MinION device

Video: MinION Introduction

Nanopore documentation: Hardware

The MinION is a portable, USB-powered device designed for DNA (or RNA) sequencing. At the core of its operation are flow cells, which serve as the functional units where sequencing occurs. Flow cells are consumable cartridges embedded with thousands of nanopores through which DNA or RNA strands pass during sequencing. These flow cells are where the sequencing is happening on the device. The MinION contains the electronics which run the flow cell, keep it at the appropriate temperature, and manage data transfer.

Technical Summary (M1kB) - Technology: Nanopore sequencing with flow cells - Flow Cells: Consumable units containing nanopores; available in various models to support different throughput needs - Sequencing Output: Up to 50 Gb per run with a standard MinION flow cell - Size: 105 mm x 23 mm x 16 mm - Weight: ~87 grams - Power Source: USB connection to a laptop or desktop computer - Software: MinKNOW for sequencing operation and basecalling; optional EPI2ME for additional analysis

MinIONs - the 'workhorse' for educational sequencing

Generally, we suggest most classroom educators will focus on MinIONs, especially as they get started with sequencing. There are other larger Nanopore sequencing platforms which offer greater data production capabilities but at a higher cost.

MinION device models: Mk1B, Mk1C, and Mk1D

Oxford Nanopore Technologies offers several models of the MinION device, each tailored to specific sequencing needs.

MinION Mk1B:

The Mk1B is a portable, USB-powered device designed for DNA and RNA sequencing. It connects to a computer via a single USB 3.0 port, providing power, temperature control, and data transfer. The device is compatible with MinION flow cells and Flongle adapters, enabling flexibility in sequencing applications.

Technical Specifications

MinION Mk1C (Discontinued):

The Mk1C integrates sequencing hardware with built-in computational capabilities, offering a compact and portable solution for nanopore sequencing. This self-contained device eliminates the need for external computing resources, as it combines sequencing and analysis within a single unit. This device is discontinued and although still functional, at some point updates to the software may be discontinued. If you have questions about this device you should contact Nanopore support directly.

Technical Specifications

MinION Mk1D:

The Mk1D features improved thermal dissipation capabilities, enhancing sequencing performance and enabling accurate real-time sequencing across diverse environments. It includes intuitive status LEDs for easy monitoring of device status. The Mk1D is compatible with MinION flow cells and Flongle adapters, supporting both DNA and RNA sequencing.

Technical Specifications

Flow cells

Flow cells are essential components of Nanopore sequencing devices. Each flow cell contains a membrane embedded with an array of nanopores. These nanopores form the core of the sequencing process by detecting the passage of nucleic acid strands through the flow cell. When DNA or RNA molecules pass through a nanopore, they cause specific disruptions in an electrical current. These disruptions are recorded and analyzed to determine the sequence of nucleotides. This real-time process enables users to generate sequencing data as the experiment progresses.

Flow cells are available in different designs to accommodate varying experimental needs.

MinION Flow Cells: These flow cells can generate up to 50 gigabases (Gb) of data in a single run, making them suitable for high-throughput applications.

MinION flow cell
Flongle Flow Cells: These are designed for smaller-scale experiments, capable of producing up to 2.8 Gb of data, offering a cost-effective option for pilot studies or limited sample sets. We recommend most educational applications strongly consider Flongles due to their lower cost (<$70 per flow cell).

Adapter required

Flongle flow cells come with an adapter that allows them to fit into the MinION devices (see figure below).

Flongle flow cell (above) and Flongel adapter (below)

Flow cell reminders

Inspect before use: Visually inspect flow cells upon receipt and before use to ensure no visible damage or contamination. Report any issues immediately to Nanopore support.
Run flow cell check before loading: Before you load your sequencing library, you can use MinKNOW software to check your flow cell and ensure it is functioning and within warranty. Contact Nanopore support for help if a flow cell does not pass. Keep in mind old or expired flow cells may still yield good-enough results, but don't depend on this when planning.
Maintain records: Keep records of purchase dates, storage conditions, and expiration dates.
Plan shipping and use promptly: Plan experiments to use flow cells as close to their purchase date as possible to maximize reliability. You can generally plan (or adjust) the ship date of your Nanopore reagents and flow cells to arrive as needed within 1 year of purchase. If you order multiple flow cells you can even break up shipments. Keep in mind that each shipment may incur its own shipping charges.

Don't freeze your flow cells - and other tips

Proper Storage and Handling of Flow Cells

Flow cells are delicate, high-precision consumables that require specific storage and handling conditions to maintain their performance. Improper storage or use can result in reduced sequencing quality or failure.

Storage and temperature requirements

Storage Temperature: Flow cells must be stored at 2°C to 8°C (refrigerated) to preserve the integrity of the nanopores and other components. They should never be frozen or exposed to temperatures outside this range.
Ensure that whomever will receive your Nanopore shipments are aware of this restriction. Your shipment may contain regents that require freezing, and it is not uncommon for an unsuspecting person in receiving or a lab manager inexperienced with Nanopore to accidentally freeze flow cells.

Shelf Life

See Flow cell warranty and storage

Flow cells have a limited shelf life, and their performance is guaranteed only within the expiration date provided by Oxford Nanopore. Always check the expiration date before use and prioritize using older flow cells first to minimize waste.
MinION shelf life: ~12 weeks
Flongle shelf life: ~4 weeks
Flow cells come with a warranty that covers manufacturing defects. However, this warranty does not apply if flow cells are damaged due to improper storage, handling, or use. Always follow the manufacturer’s guidelines to ensure eligibility for warranty claims.

Other Nanopore sequencing platforms

Video: Choosing the right Nanopore sequencing device for you

Oxford Nanopore offers a range of sequencing platforms beyond the MinION, designed for higher-throughput and more specialized applications. While these platforms provide significant sequencing capacity and flexibility, their cost and complexity make them better suited for dedicated research projects or advanced educational applications. For most classrooms and undergraduate settings, the MinION platform remains the preferred choice due to its affordability, portability, and ease of use.

GridION The GridION is a benchtop sequencing platform that can operate up to five MinION flow cells simultaneously, allowing for increased throughput. It includes integrated compute capability, enabling data analysis alongside sequencing. This device is ideal for labs requiring moderate to high-throughput sequencing in a compact setup.

Learn more about the GridION

PromethION and P2 Solo The PromethION series includes high-throughput sequencing platforms capable of handling up to 48 flow cells simultaneously in its larger models. The PromethION P2 Solo is a more compact option, supporting two PromethION flow cells, each capable of generating up to 290 gigabases (Gb) of data. These devices are suited for large-scale projects, such as sequencing entire genomes or extensive metagenomic studies.

Learn more about the PromethION series

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Library Preparation and Sequencing Kits

Core Nanopore sequencing kits

While Oxford Nanopore offers a variety of kits and applications, we generally focus on sequencing strategies that use the following three Nanopore kits.

Nanopore account required to access

Some of the links require you to sign into you Oxford Nanopore account. You may obtain a free account by signing up: Register

16S Barcoding Kit 24 V14

The 16S Barcoding Kit 24 V14 is designed for rapid and efficient amplification and sequencing of the full-length 16S rRNA gene (~1,500 bp) from extracted genomic DNA (gDNA). This kit facilitates genus-level bacterial identification by targeting the 16S rRNA gene, a highly conserved region present in all bacteria, making it a valuable tool for microbial diversity studies.

16s Workflow (as presented on Oxford Nanopore Product Page)

Key Features

Full-length amplification: Utilizes primers that amplify the entire 16S rRNA gene for accurate bacterial identification.
Multiplexing: Includes 24 unique barcodes, allowing simultaneous sequencing of up to 24 different samples in a single run, enhancing throughput and reducing costs.
Rapid library preparation: The protocol requires approximately 40 minutes plus PCR time (2-3 hours).
Minimal input requirement: Requires only 10 ng of gDNA per sample, making it suitable for samples with limited DNA quantities.
No fragmentation: Designed to work with intact gDNA without the need for fragmentation, simplifying the preparation process.

Applications

Microbial community profiling: Ideal for analyzing complex microbial populations in environmental samples, or microbiome studies.
Bacterial identification: Enables rapid identification of bacterial genera in mixed samples, aiding in diagnostics and research.
Educational applications: Suitable for teaching laboratories to demonstrate sequencing techniques and microbial diversity analysis.

What you get (and what's not included)

The product page for all Nanopore kits has a "3RD Party Materials" section that describes all of the materials and reagents not included in the kit. Review this list ahead of time to ensure you have everything you need to perform the experiment.

Rapid Barcoding Kit 24 V14

The Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) is designed for swift and straightforward preparation of up to 24 barcoded sequencing libraries without the need for PCR amplification. This kit employs a transposase enzyme to simultaneously fragment genomic DNA (gDNA) and attach unique barcoded tags, facilitating efficient multiplexing and sequencing.

Example rapid sequencing kit protocol (as presented on Oxford Nanopore Product Page). Note: Your protocol may vary based upon application.

Key Features:

PCR-Free workflow: Enables direct sequencing of gDNA without PCR amplification, preserving base modifications and reducing preparation time.
Multiplexing: Includes 24 unique barcodes, allowing simultaneous sequencing of up to 24 samples in a single run, enhancing throughput and cost-effectiveness.
Rapid library preparation: The protocol requires approximately 60 minutes, streamlining the workflow for quick results.
Minimal input: Requires only 200 ng of gDNA per sample, accommodating samples with limited DNA quantities.

To PCR or not to PCR

We also recommend this kit for DNA Barcoding (e.g. species identification using DNA); in that case, you will amplify a barcoding region (e.g., rbcL, COI) and using this kit to sequence the amplicon.

Applications:

DNA Barcoding: Barcode regions amplified by PCR or other amplicons can quickly be sequenced using this kit. Because this kit fragments PCR products, you will need to use bioinformatics to reassemble fragments.
Genomic DNA sequencing: Suitable for rapid sequencing of gDNA from various organisms, aiding in genomic studies and research.
Microbial community analysis: Facilitates the study of complex microbial populations by enabling the sequencing of multiple samples concurrently.

Ligation Sequencing Kit V14

The Ligation Sequencing Kit V14 offers a flexible method for preparing sequencing libraries from double-stranded DNA (dsDNA), including genomic DNA (gDNA), complementary DNA (cDNA), or amplicons. The straightforward library preparation involves repairing DNA ends and adding a dA-tail using the NEBNext End Repair/dA-Tailing Module, followed by ligation of sequencing adapters provided in the kit.

Ligation sequencing kit protocol (as presented on Oxford Nanopore Product Page).

Key Features:

Versatile input compatibility: Suitable for various dsDNA inputs, such as gDNA, cDNA, or amplicons, providing flexibility across different applications.
High accuracy: Optimized to deliver modal raw read accuracies over 99% (Q20+) when used with R10.4.1 nanopores, ensuring reliable sequencing data.
Long read lengths: Capable of producing read lengths equivalent to the input fragment length, facilitating comprehensive genomic analyses.
Preparation time: Approximately 120 minutes, streamlining the workflow for efficient library preparation.
Input requirement: Requires 1,000 ng of dsDNA; for fragmented or PCR-amplified DNA, 100 ng or more is sufficient, and for amplicons or cDNA, 100–200 fmol is recommended.

Applications:

Whole genome sequencing: Ideal for sequencing entire genomes, providing comprehensive coverage and high accuracy.
Transcriptome analysis: Suitable for sequencing cDNA to study gene expression and alternative splicing events.
Amplicon sequencing: Effective for sequencing PCR products, enabling targeted analysis of specific genomic regions.

Additional sequencing reagents

The sample prep product page features the entire line of kits. Some additional ones you may want to be aware of include:

Native barcoding Kit: Similar to the ligation kit, allows you to barcode your ligated products (e.g., genomic DNA, amplicons)
Flow cell wash kit: If you are using the MinION flow cell, you can use this kit to wash between uses, eliminating cross-contaminating DNA.
Sequencing auxiliary vials: During your trials or if you are doing lots of sequencing, you may eventually find you have run out of some reagents and have extra of others. These are extra vials of common reagents, allowing you to have extra on hand.
Rapid adapter auxiliary: If you are using kits with the rapid adaptor (e.g. 16s, rapid barcoding), this is the most expensive component and a limiting reagent. If you want extra, you may be able to pool and extend your kits.

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Computer and Software Management for Nanopore Sequencing

Computer Requirements

To operate a Nanopore sequencing device, selecting the right computer and software is essential for smooth sequencing runs, data management, and analysis.

The Role of Computers in Nanopore Sequencing Workflows

Computers support every stage of the sequencing process, and their use can be divided into four general functions:

To operate and control the sequencing device
The computer is responsible for running the software (e.g., MinKNOW) that controls the sequencing device and monitors the sequencing process in real time. This task does not require a high-end machine; a laptop or desktop with a quad-core processor and 16 GB of RAM is sufficient.
To provide storage for sequencing data
Sequencing experiments can generate substantial amounts of data, even with small-scale experiments producing several gigabytes. As the number and complexity of experiments grow, so do storage needs. Solid-state drives (SSDs) are recommended due to their faster read/write speeds, which are crucial for smooth operation during sequencing runs. Ample storage space (1 TB or more) ensures you can manage and retain large datasets over time but a few hundred GB should be sufficient for small-scale applications.
To perform basecalling
Basecalling is the computational process that converts raw electrical signals from the sequencer into nucleotide sequences. While this can be done on most modern computers, a well-equipped machine with a discrete GPU significantly improves processing speed and accuracy. GPUs enable the use of advanced basecalling algorithms that enhance sequence quality, particularly for high-throughput applications.
To conduct data analysis
Data analysis involves tasks such as genome assembly, variant calling, and classification of sequencing reads. While these can require substantial computational resources, many free cloud-based platforms (e.g., EPI2ME, Galaxy, or CyVerse) reduce the need for high-end hardware. As a result, lower-specification computers can still be used effectively for data analysis, particularly when combined with cloud resources.

GPUs and basecalling

What is basecalling?
Basecalling is the process of converting raw electrical signals generated during sequencing into nucleotide sequences (A, T, G, C). High-quality basecalling enhances the accuracy of downstream analyses.

Using GPUs for basecalling
While basecalling can be performed without a GPU, access to GPUs significantly accelerates the process and supports the highest-quality basecalling algorithms. Modern discrete GPUs, such as NVIDIA GeForce RTX or equivalent, improve performance dramatically.

Accessing GPUs

JetStream2 and ACCESS-CI: Free access to GPU resources is available through these academic platforms. While powerful, they require some technical expertise to set up and use effectively. Learn more about JetStream2.
Amazon Web Services (AWS): Provides scalable GPU resources for basecalling and data analysis. However, these services incur hourly costs, depending on the instance type.

No GPU? No problem!
If you do not have access to a GPU, MinKNOW includes CPU-based basecalling that is sufficient for many educational and smaller-scale projects. However, processing times will be longer, and throughput may be reduced.

Matching computer resources to workflow needs

The computer used to run the MinION can be a high-performance machine to manage device operation, data storage, and basecalling efficiently. However, student computers used for limited or cloud-based data analysis tasks don’t need to meet such demanding requirements. Cloud platforms provide cost-effective ways to leverage advanced computational tools without requiring expensive hardware, ensuring accessibility for educators and students.

Minimum System Requirements

Oxford Nanopore devices, such as the MinION Mk1B and Mk1D, have specific requirements to ensure reliable operation. At a minimum, your computer should meet the following specifications:

Operating system:
- Windows 10 or later (64-bit)
- macOS 12 Monterey or later (Apple M2/M3/M4 series or Intel processors)
Processor:
- Quad-core CPU (Intel i5/i7 or AMD equivalent) or Apple M2/M3/M4 series.
RAM:
- At least 16 GB for efficient operation
Storage:
- 500 GB SSD for data handling and storage
USB Ports:
- One USB 3.0 port for device connection
Graphics:
- Integrated graphics are sufficient for basic operations; discrete GPUs recommended for intensive tasks (see below for details on GPU use).

Warning: Do your homework - computer specifications change frequently

Please check MinION IT requirements or contact Nanopore support before making a purchase. This guide is not a replacement for doing your own homework!

Recommended Systems

For optimal performance, especially when handling large datasets or performing local basecalling, consider systems with higher specifications. Modern laptops such as the Apple MacBook Pro (M2/M3/M4 series) or high-performance Windows laptops meet these needs. The following have appeared on Nanopore educational resources as potential choices.

Apple MacBook Pro: Retail site
Razer Blade 18: Retail site

Data Storage in Nanopore Sequencing Workflows

Data storage is a critical aspect of Nanopore sequencing workflows, as even small experiments can produce several gigabytes (GB) of data. Proper storage solutions are essential for managing this data efficiently and ensuring it is secure and accessible for analysis and archiving. Both external solid-state drives (SSDs) and cloud-based storage options are options, each with advantages and limitations. Investing in appropriate storage solutions and a solid backup strategy, educators and researchers can effectively manage their sequencing data, ensuring its security, accessibility, and readiness for downstream analysis or publication.

External Solid-State Drives (SSDs)

External SSDs offer fast, reliable, and portable storage solutions, making them an excellent choice for sequencing workflows. The high read/write speeds of SSDs are especially important during sequencing runs, where real-time data transfer is required. Their durability and portability make them suitable for laboratory and fieldwork applications.

Recommendations for external SSDs

Capacity: A minimum of 500 GB is recommended for basic projects, but 1 TB or larger is ideal, especially for labs handling multiple datasets or larger-scale experiments.
Performance: Drives with high read/write speeds, such as NVMe or USB 3.2, are highly recommended to ensure efficient data handling during sequencing and analysis.

Cloud-Based storage solutions Cloud storage provides scalable and convenient options for managing sequencing data, particularly for collaborative or remote projects. Platforms like Google Drive, Dropbox, and AWS S3 allow users to upload and share data without relying on physical storage devices.

Considerations for cloud storage

Capacity and costs: While many platforms offer free storage tiers, these often have file size limits that can be restrictive for large sequencing datasets. Paid plans with higher storage capacities may be necessary as data volume increases.
Accessibility: Cloud solutions facilitate collaboration by enabling remote access and data sharing with colleagues or collaborators.
Backup: Cloud platforms can serve as a secondary or backup solution to complement local storage, ensuring data redundancy.

Data backup and archiving

For important datasets, particularly those intended for publication, a robust data backup strategy is essential. Backing up data on multiple devices or platforms minimizes the risk of data loss due to hardware failure or accidental deletion. Consider the following practices:

Use external SSDs for local backups and a cloud storage platform for off-site redundancy.
Regularly verify that backups are complete and accessible.
Implement a naming and organization strategy to ensure datasets are easy to locate and reference.

Important caveats and practices

While external and cloud-based storage solutions are effective, users should also consider:

Data privacy: Sensitive datasets, especially in clinical or ecological research, must comply with data privacy regulations.
FAIR Data management: Adhering to FAIR (Findable, Accessible, Interoperable, Reusable) principles is encouraged to promote data stewardship and reuse.

Software Requirements

Video: How to run your sequencing device and get started with data analysis

At a minimum, MinKNOW and EPI2ME software will be needed for Nanopore sequencing. In the bioinformatics section of this guide, we will provide and review additional information.

MinKNOW

MinKNOW is the primary software for operating Nanopore sequencing devices. It controls the sequencing hardware, monitors flow cell performance, and performs real-time basecalling.

Download: Available after registering an Oxford Nanopore account.
Compatibility: Pre-installed on MinION Mk1C and Mk1D; compatible with Windows and macOS systems for Mk1B and Mk1D.

EPI2ME

EPI2ME is a cloud-based platform for data analysis. It complements MinKNOW by providing tools for downstream tasks, such as read classification and genome assembly. EPI2ME is suitable for users with limited local computing resources.

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Ended: Equipment and Lab Management

Laboratory Protocols ↵

Sample Collection ↵

DNA Sampling and Extraction for Nanopore Sequencing

Introduction

Nanopore sequencing is a highly versatile technology capable of analyzing DNA directly from diverse sample types, from environmental sources to purified genomic extracts. However, the success of a sequencing experiment begins with DNA sampling and extraction—ensuring that high-quality DNA is obtained while keeping protocols accessible and cost-effective.

In this guide, we focus on DNA extraction methods suitable for classroom and teaching labs where simplicity, affordability, and safety are key considerations. We generally recommended approaches that:

Minimize toxic chemicals to ensure safety and ease of disposal.
Reduce reliance on specialized equipment to keep protocols accessible.
Lower costs by recommending practical and scalable methods.

In many cases, particularly when PCR amplification is used simpler homemade" DNA extractions can be sufficient, even when DNA integrity is not perfect. For applications that require long DNA reads such as whole-genome sequencing or structural variant analysis, we will highlight commercial extraction kits that give a better chance for high DNA integrity.

While specialized extraction methods may yield the highest-quality DNA, they often require additional steps, equipment, and reagents.

Basic principles of DNA extraction

PubMed lists more than 1600 articles on DNA extraction methods. Depending on your sample, you can find specialized methods, but we focus here on the most generalizable approaches knowing that many educators deploying in the classroom will want to focus on 1-2 methods that can cover the largest variety of samples. DNA extraction usually involves the following:

Cell lysis

A lysis buffer containing detergents, mechanical disruption, and heat are all commonly used tools. Mechanical disruption methods, such as bead beating, grinding with a mortar and pestle, or using liquid nitrogen for tough samples like plant tissue, help break open cell and nuclear membranes, allowing cellular material to be released into solution.

Protein digestion and removal

Once the cells are lysed, enzymes such as proteinase K are often added to degrade proteins that might be bound to the DNA or otherwise contaminate the sample. In addition to enzymatic digestion (e.g., lysozyme for bacterial samples), chemical agents (e.g., phenol or chloroform) can be used to separate proteins from the nucleic acids. This step helps ensure that the final DNA preparation is as free as possible from protein contaminants. We don't recommend any methods using phenol due to the increased safety hazard.

Precipitation

To isolate the DNA, an alcohol (usually ethanol or isopropanol) is added along with salts. The salts help neutralize the charges on the DNA, and the alcohol causes the DNA to precipitate out of solution. Centrifugation is then used to collect the DNA as a pellet. Most protocols will use isopropanol first since lower volumes are more efficient, and ethanol is used in washing since it has a lower evaporation point. Two important methods worth highlighting here:

Silica membrane-based purification: DNA binds to a silica column in the presence of chaotropic salts and is later eluted in water or buffer. Many commercial kits use this method and although spin columns add to the cost, column-based kits are fairly reliable and reproducible. A potential drawback is that columns may yield more fragmented DNA (i.e., DNA with lengths in the 1000's of basepairs rather than 10,000-100,000+ bp)
Magnetic bead purification: DNA attaches to magnetic beads, allowing for easy separation and cleanup. This method will frequently be used in Nanopore kits at various cleanup steps to purify DNA after enzymatic steps, rather than the initial DNA extraction.

Washing and Resuspension

The DNA pellet is washed (often with ethanol) to remove any residual salts or other impurities. After washing, the pellet is dried and then resuspended in a suitable buffer (commonly TE buffer) to stabilize the DNA for storage and subsequent applications.

Additional Reading

The Evolution of DNA Extraction Methods. Preetha J Shetty, The Evolution of DNA Extraction Methods. 2020 - 8(1). AJBSR.MS.ID.001234. DOI:10.34297/AJBSR.2020.08.001234.

DNA extraction recommendations by sample type and analysis endpoint

These are some selections we have tried and do not represent all possible or even the best possible approaches. Please give us feedback on new and/or lower-cost approaches you wish to share!

Note: All prices are estimates and may not include the total sample cost, for example including reagents or supplies not provided and/or potential education discounts.

Phage

A rapid competitive method for bacteriophage genomic DNA extraction

Format: Homemade
Sequencing endpoint: PCR amplification/16S
Costs: Low (<$1 sample)
Application notes: Protocol

Norgen Phage DNA Isolation

Format: Commercial kit
Sequencing endpoint: Whole genome
Costs: Midrange ($5 sample)
Application notes: Website

See Monarch® HMW DNA Extraction Kit for Tissue Phage protocol

Format:Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($10 per sample)
Application notes: Website

Bacteria

See Monarch® HMW DNA Extraction Kit for Tissue Bacteria protocol

Format:Commercial kit
Sequencing endpoint: Long-read whole genome or PCR amplification/16S
Costs: High ($10 per sample)
Application notes: Website

Swab collection and DNA preservation

Format: Commercial kit
Sequencing endpoint: DNA collection and preservation only (no DNA extraction)
Costs: Midrange ($7 per sample + kit for DNA extraction)
Application notes: Website

Alkaline Method for Bacterial DNA Extraction

Format: Homemade
Sequencing endpoint: PCR amplification/16S
Costs: Low (<$1 sample)
Application notes: Protocol

General microbial

Norgen Microbiome DNA Isolation Kit

Format: Commercial kit
Sequencing endpoint: PCR amplification/16S
Costs: Midrange ($6 per sample)
Application notes: Website

Soil, environmental

DNeasy PowerSoil Pro Kit

Format: Commercial kit
Sequencing endpoint: PCR amplification/16S, whole genome
Costs: High ($10 per sample)
Application notes: Website

Water, environmental

E.Z.N.A.® Water DNA Kit

Format: Commercial kit
Sequencing endpoint: PCR amplification/16S
Costs: Low ($4 per sample)
Application notes: Website

Plant

See PacBio Nanobind PanDNA kit formatted for plant nuclei lysis

Format: Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($14 per sample)
Application notes: Website

Insect

See Monarch® HMW DNA Extraction Kit for Tissue Insect protocol

Format:Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($10 per sample)
Application notes: Website

General

Simple, Robust Invertebrate DNA Barcoding: Chelex-Based DNA Extraction and Optimized COI Amplification

Format: Homemade
Sequencing endpoint: PCR amplification
Costs: Low (<$1 per sample)
Application notes: Website

Fire Monkey High Molecular Weight DNA (HMW-DNA) extraction kit

Format: Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($10 per sample)
Application notes: Website

Monarch® HMW DNA Extraction Kit for Tissue

Format:Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($10 per sample)
Application notes: Website

PacBio Nanobind PanDNA kit

Format: Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: High ($14 per sample)
Application notes: Website

New England Biolabs® (NEB) support for ORG.one participants

Format: Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: Varies
Application notes: Website

Promega Wizard® HMW DNA Extraction Kit

Format: Commercial kit
Sequencing endpoint: Long-read whole genome
Costs: Midrange ($7 per sample)
Application notes: Website

Additional Reading

Oxford Nanopore Extraction protocols page (sign-in required) https://nanoporetech.com/documentation/results?category=prepare&topic=extraction-protocols.
DNA Barcoding 101 — DNA extraction protocols. Website
New England Biolabs® (NEB) support for ORG.one participants. https://nanoporetech.com/oo/extraction-protocols.
Gand, M., Bloemen, B., Vanneste, K. et al. Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria. BMC Genomics 24, 438 (2023). https://doi.org/10.1186/s12864-023-09537-5.

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Ended: Sample Collection

Other Sequencing Protocols ↵

Ligation-based Amplicon barcoding (Long Protocol)

Summary: This detailed protocol shows how PCR amplicons can be barcoded and prepared for Nanopore sequencing.

Protocol information

Protocol Credits

Author(s)
- Luis E. Vázquez-Quiñones, Inter American University of Puerto Rico – Arecibo Campus
- Miguel Urdaneta-Colón, University of Puerto Rico - Río Piedras Campus
- José Agosto-Rivera, University of Puerto Rico - Río Piedras Campus
Maintainer/contact: Luis E. Vázquez-Quiñones, Inter American University: email
Last updated: February, 2025
Source protocols and references
- Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
- Qubit™ 1X dsDNA HS Assay

DNA source material

Type: Any double-stranded DNA
Collection source: Amplicon from PCR reaction

Nanopore Sequencing

Sequencing format: MinION OR
Sequencing kit: Native Barcoding Kit 96 V14 (SQK-NBD114.96)
Oxford Nanopore Sequencing protocol: - Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
Indexed/Barcoded: Yes, 96 indicies
Samples per run: 96 samples

Computer and Bioinformatics

Analysis tools
- Varies/ not covered in this protocol
Analysis difficulty: N/A
Command line needed: N/A
GPU/Super-high accuracy basecalling required: Varies depending on application

Reagents

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

Will vary depending on application. This protocol starts with cleaned, amplified PCR product.

DNA extraction

Not applicable. User will need to provide 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded.

DNA prep, library creation, and sequencing

Nanopore kit
- Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
  - Native Adapter
  - Sequencing Buffer
  - Library Beads OR Library solution
  - Elution buffer
  - AMPure XP beads
  - Long Fragement Buffer OR Short Fragement Buffer
  - EDTA
  - Flow Cell Flush
  - Flow Cell Tether
  - Native Barcode Plate
Qubit™ 1X dsDNA HS Assay
- Qubit™ reagents; dsDNA HS Assay Kit (ThermoFisher® Q32851)
  - Invitrogen Qubit Assay Tubes Catalog number: Q32856 0.5 mL PCR microtubes thin-wall, clear or Axygen PCR-05-C tubes (VWR, part no. 10011-830)
  - Qubit dsDNA HS Reagent
  - Qubit dsDNA HS Buffer
  - Qubit dilution buffer
User-provided reagents
- NEB Blunt/TA Ligase Master Mix (NEB, cat No. M0367)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, cat No. E7546)
- NEBNext Quick Ligation Module (NEB, cat No. E6056)
- Agencourt AMPure XP beads
- Nuclease-free water (e.g. ThermoFisher, cat No. AM9937)
- Freshly prepared 80% ethanol in nuclease-free water (500 µL per PCR reaction to be cleaned up)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Equipment and consumables

Lab equipment

Micropipette set (e.g., P10, P100, P1000) and tips
Microcentrifuge (20,000 x g)
Heat block or water bath
Qubit (dsDNA HS Assay Kit - ThermoFisher, Q32851; and assay tubes) or Nanodrop spectrophotometer for DNA quantification
Permanent markers
Magnetic rack for microfugue tubes(1)
PCR machine (for incubation of samples in PCR tubes)
Vortex mixer

Consumables

Assorted tube racks (microfuge, conical tubes, and PCR tubes)
1.5-1.7ml microfugue tubes
0.2 ml thin-walled PCR tubes
(Optional) 1.5 ml Eppendorf DNA LoBind tubes

Nanopore sequencing equipment

Sequencing device: MinION or Flongle w/Adaptor

Computer equipment

Desktop or laptop

Recommendation - Sergi Lab Supplies

Estimated timings

DNA End preperation: 30-45 mins

Stop-point: Store the samples overnight at 4 °C.

Barcode ligation: time 60-90 mins

Stop-point: Store the samples overnight at 4 °C

Adapter ligation: 60-70 mins

Stop-point: Store the samples overnight at 4 °C or long-term at -80 °C.

Flow cell loading: 20-30 mins

Background

The ONT Ligation Sequencing procedure is a method of preparing DNA molecules for sequencing in a Nanopore system using different dsDNA sources. It is a flexible method that can be used with gDNA, cDNA, or amplicons. The throughput is higher than the ONT Rapid methods but is more expensive because of reagents from other companies and has a longer time for preparation. Native Barcoding with ligation is a method for multiplex samples to reduce the cost of the samples. Multiplexing may be used only when the amount of data required is less than the total of data that can be generated from a single flow cell.

The preparation of the sample has four principal steps:

the DNA end preparation,
the Barcode ligation,
the adapter ligation and
loading the flow cell.

Additional Reading

Some reading and potentially links to references.

Library Preperation - Native Barcoding Prep (96)

Goal: Attach sequencing barcodes and adaptors necessary for nanopore sequencing.

Nanopore

This protocol follows the MinION version of Native Barcoding Kit 96 V14 (SQK-NBD114.96) from Oxford Nanopore.

Preliminary Steps

Goal: Ensure DNA ends are enzymatically prepared to attach barcodes.

End-repair

1. Add 200 fmol (130 ng for 1 kb amplicons) of each sample into a clean 96-well microplate (if you have 96 samples or prepare microtubes for the number of samples available).
2. Complete the volume of each sample up to 11.5 μL with nuclease-free water.
3. Pipette to mix gently, and spin down afterwards.
4. In each well or microtube, mix the following components.
✓ Ingredient Volume
200 fmol Amplicon DNA

11.5 µL
Diluted DNA Control Sample (DCS)

1 µL
Ultra II End-prep Reaction Buffer

1.75 μL
Ultra II End-prep Enzyme Mix

0.75 μL
5. Make sure all the ingredients are well/microtube combined by pipetting and briefly spinning them down.
6. In a thermal cycler Incubate for five minutes at 20 °C and five minutes at 65 °C.

Native Barcode Ligation

Preliminary steps

Note

Refer to the manufacturer's instructions to prepare the NEB Blunt/TA Master Mix and place it on ice.

1. Thaw all the reagents at room temperature.
2. Spin down the tubes containing the reagent for 5 seconds.
3. Perform 10 full volume pipette mixes to make sure that all reagents are thoroughly mixed.
- a. Thaw the AMpure XP Beads (AXP) at room temperature and then mix with a vortex. Then keep them at room temperature.
- b. Thaw the EDTA at room temperature and then mix with a vortex, spin down, and then place on ice.
- c. According to the number of samples to be used, select the number of Native Barcodes from NB01 ![][image4] to NB96 and thaw at room temperature.
  
  Note
  
  Individually mix the barcodes by pipetting, spin down, and then place on ice.
- d. For every individual sample select a unique barcode.
  
  Note
  
  At the final steps of the preparation, you will mix all the barcoded samples and run on the same flow cell.
- e. Using a new 96 well place add the reagents in the following order.
  ✓ Ingredient Volume
  Nuclease free water 3 µL
  End-prepared DNA 0.75 µL
  Individual Native Barcode 1.25 μL
  Blunt/TA Ligase Master Mix 5 μL

Pause point

At this step, you may stop the procedure and store the sample overnight at 4°C.

Instructions
- a. Mix the reaction thoroughly by gently pipetting and briefly spinning down.
- b. Incubate at room temperature for 20 minutes.
2. To each well or 0.2 mL microtube, add the following volume:
✓ Clear cap EDTA Blue cap EDTA
1 µL

or

2 µL
3. Then mix thoroughly by pipetting and spin down briefly.
4. Pool all individual samples and purify them with the AMPure Beads on a magnetic rack.
- a. Pool all the individual samples into one 1.5 mL Eppendorf DNA LoBind microtube.
- b. Resuspend by vortexing the AMPure XP Beads (AXP).
- c. Add 0.4X AMPure XP Beads (AXP) beads to the Pooled Reaction then mix by pipetting.
- d. Mix on a rotator mixer (Hula Mixer or other) for 10 minutes at room temperature.
- e. During the mix step prepare 2 mL of 80% ethanol in Nuclease-free water.
- f. When finished with the mix, spin down the sample and put it on a magnetic rack to pellet for 5 minutes.
  
  Note
  
  You will notice that the liquid turns clear, and the pellet is aside on the magnet.
- g. Without removing the microtube from the magnetic rack carefully remove the clear portion with a pipette without disturbing the pellet.
- h. Do not remove the tube from the magnetic rack and wash without disturbing the pellet with 700 µL of the freshly prepared 80% ethanol.
- i. With the tube on the rack carefully remove and discard the ethanol.
- j. Repeat the previous step to remove the ethanol.
- k. Take out the microtube from the rack and spin it down.
- l. Place back the tube on the magnetic rack and pipette out any alcohol.
- m. Allow the pellet to dry for approximately 30 seconds.
  
  Note
  
  Move quickly to avoid excess drying that can crack the beads.
- n. Take out the microtube from the magnetic rack and add 35 µL of Nuclease-free water.
- o. Gently flick the tube with your finger.
- p. Incubate the sample for 10 minutes at 37 °C and agitate every 2 minutes by gently flicking the tube with your finger.
- q. Put the sample back on the magnetic rack until the eluate is completely clear.
- r. Without disturbing the pellet, remove and retain the clear eluate in a new, clean 1.5 mL Eppendorf DNA LoBind microtube.
- s. DNA quantification: Quantify the sample using a Qubit® Fluorometer, Nanodrop®, QIAxpert® or other.

Pause point

At this step, you may stop the procedure and store the sample overnight at 4°C.

Adapter ligation and clean-up

Adapter ligation

Important

Do not interchange the Native Adapter (NA) of this kit with another sequencing adapter.

Prepare the NEBNext Quick Ligation Reaction Module according to the instructions of the manufacturer.

1. Thaw the following reagents at room temperature:
- a. Native Adapter (NA)
- b. Quick T4 DNA Ligase
- c. NEBNext Quick Ligation Reaction
- d. Elution Buffer (EB)
Mix by vortexing.
- e. Short Fragment Buffer (SFB) ; for all sizes DNA fragments or Long Fragments Buffer (LFB) for fragments of 3 kb or longer.

Important

The Quick T4 DNA Ligase should not be vortexed.

2. Spin down all reagents in a microcentrifuge for 5 seconds.
3. Mix thoroughly the reagents by pipetting up and down 10 times.

Important

The Quick T4 DNA Ligase should not be vortexed.

4. The Native Adapter (NA) and Quick T4 DNA Ligase must be first spin down, followed by pipette mixing and afterwards placed on ice.
5. Mix thoroughly the Elution Buffer (EB) ![A screenshot of a computerDescription automatically generated][image1] by vortexing, spin down, and place on ice.

Important

The clean-up step following adaptor ligation is intended to either enrich DNA segments of > 3 kb or purify all fragments equally, depending on the wash buffer (LFB or SFB) utilized. Use Long Fragment Buffer (LFB) to enrich DNA pieces 3 kb or longer. Use Short Fragment Buffer (SFB).

6. Mix in the following order these reagents in a 1.5 mL Eppendorf LoBind tube.
✓ Ingredient Volume
Pooled barcoded sample

30 µL
Native Adapter (NA)

5 µL
NEBNext Quick Ligation Reaction (5X)

10 µL
Quick T4 DNA Ligase

5 µL
7. Spinning down briefly.
8. Incubate for 20 minutes at Room Temperature.

Clean-up using the AMPure XP Beads

Important

This procedure uses (LFB) or (SFB) but not 80% ethanol to wash the beads because ethanol will be detrimental to the sequencing reaction. The sequencing reaction will be hampered if ethanol is used.

Important

It is recommended to load 10-20 fmol of the final prepared library onto the R10.4.1 ONT MinION flow cell (FLO-MIN114). The final prepared library should be stored on ice until ready for loading.

DNA quantification

1. Quantify the sample using a Qubit™ Fluorometer (Invitrogen™, Q32856), Nanodrop™, QIAxpert® or other for DNA quantification.

Steps to quantify the DNA

1. Set aside two microtubes for the standards (dsDNA High Sensitivity needs 2 standards) and one microtube per sample.
2. Label the microtubes on the lid (DO NOT label on the side), the instrument will be reading the sample through the microtube side.
3. The working solution needs to be prepared in a plastic tube diluting the dsDNA HS reagent 1:200 in dsDNA HS Buffer.
- a. Now use your Qubit reagent calculator and indicate the number of samples & 2 standards.
- b. Prepare the Qubit working solution by mixing the volume of Qubit dye and the volume of Qubit dilution.

Important

Do not mix the working solution in a glass container.

*Figure 1: Qubit home screen: activate the Reagent Calculator button*This image shows the Qubit home screen in which the user can choose an assay (dsDNA, RNA, Oligo, Protein, Ion Sphere, and Fluorometer). In addition, at the bottom of the screen in the middle part, the Reagent Calculator is located, and we need to activate it to compute the reagent recipe that we need to quantify our samples.

*Figure 2: Qubit Reagent Calculator*This image shows the Qubit Reagent Calculator screen: The user needs to input the number of samples in the first field, then the user inputs the number of standards in the second field. As the third step, the user needs to check the box to include overage (1 extra tube). Once the user inputs the above information, the results for the recipe appear on screen to prepare the reagent. Once the user writes down the recipe on a piece of paper, click on the Done button to return to the main screen.
(2-1) Type the number of samples, (2-2) Type the number of standards, (2-3) Select include overage, (2-4) Use the results to prepare the reagent, and (2-5) Active the Done button when finished

4. Add the working solution to the microtubes:
- Standards
  - 190 µL of the working solution per microtube
- Samples
  - 199 µL of the working solution per microtube
5. Add the samples:
- Standards
  - 10 µL of the standard to their corresponding microtube and mix it by vortexing for 2–3 seconds to avoid creating bubbles.
- Samples
  - 1 µL of the standard into their corresponding microtube and mix it by vortexing for 2–3 seconds to avoid creating bubbles.
6. Incubate the samples and standards for 2 minutes at room temperature.
7. Now select the assay type dsDNA HS (High Sensitivity; Figure 3-4).

*Figure 3: Qubit home screen: Choose an assay - dsDNA*This image shows the Qubit home screen so the user can select an assay in our case we need to select dsDNA.

*Figure 4: Qubit: choose an assay screen: dsDNA - High sensitivity*This image shows the Qubit screen so the user can select dsDNA: High sensitivity.

8. Read the standards (Figure 5)

*Figure 5: dsDNA — High sensitivity screen: Read the standards.*This image shows the Qubit screen to activate the button to read the standards.

*Figure 6: dsDNA: High sensitivity screen: Read Standard 1*This image shows the Qubit screen instructing the user to insert standard 1 and activate the read standard button and then insert standard 2 and activate the read standard button.

a. Read standard 2.
9. Now, read the samples and start preparing the pooled sample.

Example

Example: Quantifying DNA with the Qubit

Sample	[DNA] (ng/µL)
1	32.8
2	29.6
3	41.2
4	44.0
5	41.6
6	27.4

☐ Identify the sample with the highest [DNA].
☐ Divide the highest concentration by the concentration of each sample to obtain the volume that needs to be transferred for the pool.

Sample	[DNA] (ng/µL)	Volume for pooling the samples
1	32.8	44.0 / 32.8 \= 1.34 µL \= 1.3 µL
2	29.6	44.0 / 29.6 \= 1.49 µL \= 1.5 µL
3	41.2	44.0 / 41.2 \= 1.07 µL \= 1.1 µL
4	44.0	44.0 / 44.0 \= 1.00 µL \= 1.0 µL
5	41.6	44.0 / 41.6 \= 1.06 µL \= 1.1 µL
6	27.4	44.0 / 27.4 \= 1.61 µL \= 1.6 µL
	Total	7.6 µL

☐ Compute the total mass
Total DNA ng = Total volume in the pooled sample (µL) x [Highest DNA] (ng/µL)
Total DNA ng = 7.6 µL x 44.0 ng/µL = 334.4 ng

10. Connect to the NEBioCalculator and compute the fmol for your pooled samples.

Figure 7: Computing the femtomoles of DNA in our pooled sampleThis image shows the New England Biolabs Calculator to compute the double stranded DNA mass to moles:Step 1: Select ds: Mass to Moles or viceversa.Step 2:2a) Type the DNA length.2b) Select the DNA length units.Step 3:3a) Type the DNA mass.3b) Select the DNA mass units.Step 4: Now you have the fmol of DNA in your pooled sample.
- a. Select ds: Mass ⇄ Moles
- b. Type the DNA length; you need to determine the average length of the fragments to compute the fmoles. In our case, the average length for our amplicon is 1,500 bp.
- c. Select the DNA length units.
- d. Type the DNA mass.
- e. Select the DNA mass units; Now you have the fmol of DNA in your pooled sample.
11. Compute the pooled samples volume that we need to transfer to the sample for sequencing.

$\frac{12 µL}{X µL} = \frac{361.9 fmol}{20 fmol}$
$240 = 361.9 x$
$\frac{240}{361.9} = \frac{361.9 x}{361.9}$
$x = 0.66 µL$

Priming and loading the SpotON flow cell

Important

The “Ligation sequencing amplicons – Native Barcoding Kit 96 V14 (SQK-NBD114.96)” is only compatible with R10.4.1 flow cells (FLO-MIN114).

Preliminary steps

Before combining by vortexing, thaw at room temperature the following reagents spin them down once thawed and store them on ice.

✓	Reagents
	Sequencing Buffer (SB)
	One tube of Flow Cell Flush (FCF)
	Library Beads (LIB) for most experiments or Library Solution (LIS)
	Flow Cell Tether (FCT)
	50 mg/mL Bovine Serum Albumin (BSA)

Important

To get the best sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), it is recommended to add Bovine Serum Albumin (BSA) to the flow cell priming mix to a final concentration of 0.2 mg/mL. BSA ensures that more DNA strands are available for the nanopores by blocking non-specific binding in the flow cell, which boosts sequencing output.

1. Combine the following reagents to prepare the flow cell priming mix with BSA and mix by inverting the tube and pipette mix at room temperature.

Ingredient	Vol. (+ BSA)	Vol. (- BSA)
Flow Cell Flush (FCF)	1,170 µL	1,170 µL
50 mg/mL Bovine Serum Albumin (BSA)	5 µL
Nuclease-free water		5 µL
Flow Cell Tether (FCT)	30 µL	30 µL
Total	1,205 µL	1,205 µL

Note

BSA ensures that more DNA strands are available for the nanopores by blocking non-specific binding in the flow cell, which boosts sequencing output.
his solution contains a hydrophobic tether molecule used to anchor the nucleic acid onto the membrane close to the nanopore.

2. Slide the flow cell inside under the MinION’s clip by opening its lid; to ensure correct thermal and electrical contact press down firmly the flow cell.

Important

When removing the transport buffer from the flow cell, proceed with caution. Set your P-1000 micropipette to 500 µL and remove 20-30 µL of the transportation buffer slowly by only using the piston of your micropipette. Removing more than 20–30 μL is not advised. It is of the utmost importance that the buffer always covers the array of pores. Pores might sustain irreversible damage when air bubbles are introduced into the array.

Ending the experiment

1. Stop the sequencing by clicking on the stop button.
2. If you would like to reuse the flow cell after your sequencing experiment is over, please follow the instructions in the Flow Cell Wash Kit and keep the cleaned flow cell at 2-8 °C.
3. Alternatively, clean out the flow cell and prepare it for return to Oxford Nanopore by following the returns protocol.

Comments and discussion

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Ended: Other Sequencing Protocols

Ended: Laboratory Protocols

Annotated Experiments ↵

Curated Experiment: DNA Barcoding

Identifying Taxa through DNA barocode sequences

Summary: Identifying species by sequencing "DNA Barcodes" and compare sequences to a reference library.

Protocol information

Protocol Credits

Author(s)
- Anna Feitzinger, Cold Spring Harbor Laboratory
- Jeffry Petracca, Cold Spring Harbor Laboratory
Maintainer/contact: Anna Feitzinger, Cold Spring Harbor Laboratory: email
Last updated: March, 2025
Source materials and references
- Barcoding 101 Lab Resources
- Rapid barcoding protocol

Nanopore Sequencing

Sequencing format: Flongle
Sequencing kit: Rapid Barcoding Kit 24 v14 (SQK-RBK114.24)
Oxford Nanopore Sequencing protocol: Rapid Barcoding Kit (SQK-RBK114.24)
Indexed/Barcoded: Yes, 24 indicies
Samples per run: 24 samples

Computer and Bioinformatics

Analysis tools
- EPI2ME
  - EPI2ME amplicon workflow
Software to download or install:
- Oxford Nanopore Software Downloads Oxford Nanopore account and login required:
  - MinKNOW
  - EPI2ME Desktop Application
- Other software:
  - Docker
Analysis difficulty: Easier
Command line needed: No
GPU/Super-high accuracy basecalling required: No

Reagents

Sample collection and prep

See resources and recipies at barcoding101 Sample Collections

DNA extraction

For rapid DNA extraction only:

Lysis solution (6 M Guanidine Hydrochloride GuHCl) (120 µL)
Wash buffer (480 µL)
TE buffer (75 µL)
Specimen tissue sample(s)
2 Sterile plastic pestles
Whatman No.1 Chromatography paper discs (2, 3-mm diameter)

For chelex DNA extraction only:

10% Chelex Solution (2 tubes of 100 µL)
2 Sterile plastic pestles
6 Sterile toothpicks or pipette tips
Tissue sample(s) (from Part I)
2 Microcentrifuge tubes (1.5mL)

PCR

For Ready-To-Go PCR Bead Amplification:
- 2 Ready-To-Go PCR Beads in 0.2- or 0.5-mL PCR tubes
- Appropriate primer/loading dye mix (50 µL; 23 µL per reaction)*

Barcoding Primers

Plant Primers:

rbcLa Forward 5’- TGTAAAACGACGGCCAGTATGTCACCACAAACAGAGACTAAAGC-3’
rbcLa reverse 5’- CAGGAAACAGCTATGACGTAAAATCAAGTCCACCRCG-3’

Invertebrate Primers:

LCO1490 5'-TGTAAAACGACGGCCAGTGGTCAACAAATCATAAAGATATTGG-3'
HC02198 5'-CAGGAAACAGCTATGACTAAACTTCAGGGTGACCAAAAAATCA-3'

Fungi Primers:

ITS1F 5'-TGTAAAACGACGGCCAGTTCCGTAGGTGAACCTGCGG-3'
ITS4 5'-CAGGAAACAGCTATGACTCCTCCGCTTATTGATATGC-3'

DNA prep, library creation, and sequencing

Rapid Barcoding Kit (SQK-RBK114.24)
Flongle Expansion Kit (EXP-FLP002)
Flongle Flow Cell (FLG-FLO114)

Equipment and consumables

Lab equipment

Micropipettes and tips (2-1000 μL)
Magnetic microfuge tube racks
Microfuge tube rack
Microcentrifuge
Thermal cycler
Qubit™ tubes

Consumables

1.5 mL microfuge tubes
PCR tubes
Qubit™ fluorometer, Nanodrop™, QIAxpert®, or other method of DNA quantification

Nanopore sequencing equipment

MinION Flongle Adapter
MinION Mk1B or Mk1D sequencer

Computer equipment

Desktop or laptop with MinKNOW software

Estimated timings

Sample Collection: Variable
DNA extraction: 60-75 minutes
PCR, DNA quality control, and library prep:
- PCR: ~2 hours
- Quality control: 10 min (Qubit); 60-75 min (electrophoresis)
- Library prep: 30-60 min
Sequencing: Atleast 60 min
Data analysis: 30-60 min

Background

Identifying organisms has grown in importance as we monitor the biological effects of global climate change and attempt to preserve species diversity in the face of accelerating habitat destruction. We know very little about the diversity of plants and animals—let alone microbes—living in many unique ecosystems on earth. Less than two million of the estimated 5-50 million plant and animal species have been identified. Scientists agree that the yearly rate of extinction has increased from about one species per million to 100-1,000 per million. This means that thousands of plants and animals are lost each year. Most of these have not yet been identified.

Classical taxonomy falls short in this race to catalog biological diversity before it disappears. Specimens must be carefully collected and handled to preserve their distinguishing features. Differentiating subtle anatomical differences between closely related species requires the subjective judgment of a highly trained specialist – and few are being produced in colleges today.

DNA barcodes allow non-experts to objectively identify species – even from small, damaged, or industrially processed material. Just as the unique pattern of bars in a universal product code (UPC) identifies each consumer product, a “DNA barcode” is a unique pattern of DNA sequence that can potentially identify each living thing. Short DNA barcodes, about 700 nucleotides in length, can be quickly processed from thousands of specimens and unambiguously analyzed by computer programs.

DNA amplicons are the copied sections of DNA produced by an amplification process that replicates a particμLar target region of a DNA molecule. For example, DNA amplicons are commonly generated via the polymerase chain reaction (PCR).

In DNA barcoding, a DNA amplicon might represent a region of DNA that can be used to determine the species of an organism. In medical research, amplicon DNA sequences might be used to detect pathogens, search for inherited diseases, or identify single nucleotide polymorphisms (SNPs) that are associated with a particular disease phenotype. More broadly, amplicon sequencing can be used to assess and analyze genetic variation at particular positions in a genome.

Explanation

Because the regions of DNA used for identification are approximately ~70% conversved, differences can be used to distinguish between species but allows the same primer pair to be use across many species.

rbcL or matK located on the chloroplast are used to identify plants
CO1 located on the mitochondria is used for animals, including invertebrates.
ITS located in nuclear ribosomal DNA (rDNA) is used for fungi

DNA sequencing can be carried out in a number of different ways depending on the needs of an experiment. The ONT MinION sequencing device offers a portable, versatile, efficient, and relatively inexpensive way to sequence DNA in a classroom setting. In order to sequence DNA amplicons on this platform, they must first be prepared using an ONT kit. One efficient option for amplicons is the Rapid Barcoding Kit, which we used in this protocol.

Example Projects

DNA Barcoding Student Project Examples

Example of DNA Barcoding student projects can be found for the DNALC's barcoding programs below:

Additional Reading

Hebert P.D., Cywinska A., Ball S.L., deWaard J.R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270(1512): 313-21.
Hebert P.D.N., Penton E.H., Burns J.M., Janzen D.H., Hallwachs W. (2004). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA. 101(41):14812-7.
Hollingsworth P.M. et al (2009). A DNA barcode for land plants. Proc Natl Acad Sci USA 106(31): 12794-7.
Ratnasingham, S., Hebert, P.D.N (2007). BOLD: The Barcode of Life Data System. Molecular Ecology Notes 7(3): 355-64.
Stoeckle M. (2003). Taxonomy, DNA, and the Bar Code of Life. BioScience 53(9): 2-3.
Van Den Berg C., Higgins W.E., Dressler R.L., Whitten W.M., Soto-Arenas M.A., Chase M.W. (2009) A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences. Annals of Botany 104(3): 417-30.
Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I, Lipman D.J., Ostell J., Sayers E.W. (2013). Nucleic Acids Res. GenBank. 41(D1): D36–D42.

Sample Collection

Goal: Collect samples for DNA Barcoding

Instruction tip

Each student (up to 24) can prepare their own sample.

Summary

Your collection of specimens may support a census of life in a specific area or habitat, an evaluation of products purchased in restaurants or supermarkets, or may contribute to a larger “campaign” to assess biodiversity across large areas. It may make sense for you to use sampling techniques from ecology. For example, a quadrat samples the plant and/or animal life in one square meter (or ¼ square meter) of habitat, while a transect collects samples along a fixed path through a habitat. A “Hula Hoop” can be used as an acceptable substitute for a quadrat.Do not take more sample than you need. Only a small amount of tissue is needed for DNA extraction—a piece of plant leaf about ⅛- to ¼-inch diameter or a piece of animal or fungal the size of a grain of rice.

Minimize damage to living plants by collecting a single leaf or bud, or several needles. When possible, use young, fresh leaves or buds. Flexible, non-waxy leaves work best. Tougher materials, such as pine needles or holly leaves, can work if the sample is kept small and is ground well. Dormant leaf buds can often be obtained from bushes and trees that have dropped leaves. Fresh, frozen leaves work well. Dried leaves and herbarium samples are variable.

Avoid twigs or bark. If woody material must be used, select flexible twigs with soft pith inside. As a last resort, scrape a small sample of the softer, growing cambium just beneath the bark. Roots and tubers are a poor choice, because high concentrations of storage starches and other sugars can interfere with DNA extraction.

DNA Extraction

Goal: Extract DNA from tissue samples

Summary

The Chelex, Silica, and Rapid protocol are all simple and efficient DNA extraction methods suitable for the classroom.

The Rapid DNA extraction protocol works extremely well for plants and for select groups of terrestrial invertebrates. This method eliminates the need for equipment such as water baths and centrifuges, and students can complete the DNA isolation and have DNA ready for PCR in <30 min.
The Chelex DNA extraction uses chelex resin which binds to substances that can inhibit PCR (polymerase chain reaction, a common DNA analysis technique), like metal ions. The DNA is then released from the sample by heating.
The Silica DNA extraction method is inexpensive and has the advantage of working reproducibly with almost any kind of plant, fungus, or animal specimen.

More information about the percentage of success of theses DNA extraction methods for different organism types can be found under the "Isolation DNA" section in here.

Pre-isolation preparation: If sample has been stored in 95%+ EtOH prior to DNA isolation, remove the sample from the EtOH using a sterile toothpick and allow it to dry for >10 min on a clean surface. Proceed when sample no longer smells of EtOH. Ethanol carried through the DNA isolation can inhibit DNA amplification later.

Rapid ExtractionChelexSilica

Steps:

Obtain tissue ~10 mg or ⅛- to ¼-inch diameter in size by removing a piece of the tissue with a razor blade or sterile tweezer. Be sure to preserve remainder of the organism, as well as additional collected specimens, at -20°C, in 95%+ EtOH, or both. Place tissue in a clean 1.5-mL tube labeled with a sample identification number.
Add 50 µL of lysis solution to each tube.

Explanation

Lysis solution dissolves membrane-bound organelles including the nucleus, mitochondria, and chloroplast..

Twist a clean plastic pestle against the inner surface of 1.5-mL tube to forcefully grind the tissue for at least 2 minutes. Use a clean pestle for each sample. Ensure the sample is ground into fine particles.

Explanation

Grinding breaks up cell walls and other tough material. Once ground, the sample should be liquid, but there may be some particulate matter remaining.

For each sample, use a separate sterile tweezer to add one 3-mm diameter disc of Whatman No. 1 Chromatography paper to the lysed extract. Tap or flick the tube gently to ensure the disc is fully submerged in the extract. Allow the disc to soak in the extract for 1 minute.

Explanation

Whatman chromatography paper binds the DNA, helping separate DNA from contaminants.

While the disc is soaking, add 200 µL of wash buffer to a clean 1.5-mL tube labeled with the sample identification number. Wash buffer will remove contaminants that can inhibit PCR while the DNA remains bound to the paper.
Remove the disc from the extract using a sterile tweezer or pipette tip and transfer the disc into the fresh tube containing wash buffer. Tap or flick the tube to mix for 5 seconds, then allow the disc to sit in the wash buffer for 1 minute. Discard/set-aside the tweezer following Step 7. Use of the tweezer to transfer the disc in future steps will contaminate the disc with impurities that may affect PCR.
Use a sterile pipette tip to gently drag the disc out of the wash buffer and up the tube wall to dry at the top of the tube. Ensure that little to no debris is attached to the disk. Allow the disc to air dry for 2 minutes to evaporate the ethanol on the disc.

Explanation

Ethanol in the wash buffer can inhibit PCR, so drying the paper after the wash step is required.

While the disc is air-drying, add 30 µL of TE to a clean 1.5-mL tube labeled with the sample identification number.
Once dry, carefully transfer the disc using a sterile tweezer or pipette tip into the fresh tube containing 30 µL of TE. Allow the disc to soak for a minimum of 15 minutes at ambient temperature (soaking the disc overnight at 4° C is optimal) to elute the purified DNA
The disc in TE can be stored at 4° C temporarily or frozen at -20° C for long-term storage until ready to begin Part III; ensure that the disc has incubated at ambient temperature for at least 15 minutes before storage at 4° C or -20° C. In Part III, you will use 2 μL of DNA for each PCR reaction. This is a crude DNA extract and contains nucleases that will eventually fragment the DNA at room temperature. Keep the sample cold to limit this activity.

Steps:

Obtain tissue ~10 mg or ⅛- to ¼-inch diameter in size by removing a piece of the tissue with a razor blade or sterile tweezer. Some organisms or samples will be small enough that the entire specimen should be used. If you are working with more than one sample, be careful not to cross-contaminate specimens. Be sure to preserve remainder of the organism, as well as additional collected specimens, at -20°C, in 95%+ EtOH, or both.

Explanation

Tissue should be no larger than a grain of rice. Using more than the recommended amount can inhibit amplification.

Gently tap 10% Chelex solution tube on a hard surface to ensure the solution is at the bottom. Place tissue into Chelex tube labeled with a sample identification number.

Explanation

Chelex binds positively charged contaminants that could be inhibitors of DNA amplification and binds magnesium ions that are cofactors of DNA nucleases.

Twist a clean plastic pestle against the inner surface of Chelex tube to forcefully grind the tissue for at least 2 minutes. Use a clean pestle for each sample. Ensure the sample is ground into fine particles. Securely close the cap of the tube.

Explanation

Grinding breaks up cell walls and other tough material. Once ground, the sample should be liquid, but there may be some particulate matter remaining.

Fill a mug nearly to the top with boiling water and cover with aluminum foil. Punch small guide holes in the foil for the number of samples you are processing. Prevent the tube from opening in the following step by using a cap lock to secure the cap to the rim of the tube. Be sure that both the tube rim and cap are held within the cap lock so that steam can’t force the cap open. Place tubes through foil so that the Chelex and sample mixture is fully submerged, but do not submerge the top of the tube.

Explanation

The heat will help to lyse cell and organelle membranes, releasing the DNA.

Incubate tubes for 10 minutes. It is not necessary to keep water boiling. Alternatively, you can use a water bath or thermocycler to incubate tubes at 95°C for 10 minutes. Discard water from mug and return the tubes in foil to the mug. Allow Chelex to settle for an additional 10 minutes. Alternatively, place tubes in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet Chelex. Once boiled, the DNA and Chelex mixture can be stored at 4° C temporarily, frozen at -20° C for long-term storage, or shipped at ambient temperature.
Carefully transfer ~30 µL of supernatant from the Chelex tube, avoiding the Chelex, into a clean 1.5mL microcentrifuge tube labelled with the sample identification number. This tube can be stored at 4° C temporarily or frozen at -20° C for long-term storage until ready to begin Part III. Alternatively, 2 µL of the supernatant from the Chelex tube can be used directly for future PCR reactions. Do not transfer any of the white Chelex resin along with the supernatant. This is a crude DNA extract and contains nucleases that will eventually fragment the DNA at room temperature. Keep the sample cold to limit this activity. Chelex inhibits DNA amplification by PCR.

Steps:

Obtain plant, fungal, or animal tissue ~10 mg or ⅛- to ¼-inch diameter by removing a piece of the tissue with a razor blade, clean tweezers, scissors, or back of a 10-µL pipette tip to enable efficient lysis. If you are working with more than one sample, be careful not to cross-contaminate specimens. (If you only have one specimen, make a balance tube with the appropriate volume of water for centrifugation steps.)

Explanation

Tissue should be no larger than a grain of rice. Using more than the recommended amount can affect amplification.

Place sample in a clean 1.5-mL tube labeled with an identification number.
Add 300 µL of lysis solution to each tube.

Explanation

Lysis solution dissolves membrane-bound organelles including the nucleus, mitochondria, and chloroplast.

Twist a clean plastic pestle against the inner surface of the 1.5-mL tube to forcefully grind the tissue for 2 minutes. Use a clean pestle for each tube if you are doing more than one sample.

Explanation

Grinding the tissue breaks up cell walls and other tough material. Once fully ground, the sample should be liquid, but there may be some particulate matter remaining.

Incubate the tube in a water bath or heat block at 65° C for 10 minutes.
Place your tube and those of other groups in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for one minute at maximum speed to pellet debris.
Label a clean 1.5-mL tube with your sample number. Transfer 150 μL of the supernatant (clear solution above pellet at bottom of tube) to the fresh tube. Be careful not to disturb the debris pellet when transferring the supernatant. Discard old tube containing the debris.
Add 3 μL of silica resin to tube; ensure silica resin is mixed and homogenous. Close tube and mix well by flicking or vortexing (solution will turn cloudy, but silica will settle shortly after). Close and incubate the tube for 5 minutes in a water bath or heat block at 57° C.

Explanation

Silica resin is a DNA binding matrix that is white. In the presence of the lysis solution the silica resin binds readily to nucleic acids.

Place your tube and those of other groups in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the resin. Use a micropipette with fresh tip to remove all supernatant, being careful not to disrupt the white silica resin pellet at the bottom of the tube.

Explanation

Centrifugation pellets the silica resin, which is now bound to nucleic acid. The pellet will appear as a tiny teardrop-shaped smear or particles on the bottom side of the tube underneath the hinge.

Add 500 μL of ice cold wash buffer to the pellet. Mix well by pipetting up and down (or by closing the tube and flicking or vortexing) to resuspend the silica resin.

Explanation

Wash buffer removes contaminants from the sample while nucleic acids remain bound to the resin. The silica resin is not soluble in the wash buffer. The silica resin may stay as a pellet or break up during the washing.

Place your tube and those of other groups in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the resin. Use a micropipette with fresh tip to remove all supernatant, being careful not to disrupt the white silica resin pellet at the bottom of the tube.
Once again, add 500 μL of ice cold wash buffer to the pellet. Close tube and mix well by vortexing or by pipetting up and down to resuspend the silica resin.

Explanation

Washing twice is much more effective than washing once with twice the volume.

Place your tube and those of other groups in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the silica resin.
There will be approximately 50 μL of supernatant remaining after the brief spin to be removed. In the presence of water or TE buffer, nucleic acids are eluted from the silica resin.
Use a micropipette with fresh tip to remove the supernatant, being careful not to disrupt the white pellet at the bottom of the tube. Spin the tube again for ~15 seconds to collect any drops of supernatant and then remove these with a micropipette.
Add 100 μL of distilled water (or TE buffer) to the silica resin and mix well by vortexing or by pipetting up and down. Incubate the mixture at 57° C for 5 minutes.
For long-term storage it is recommended DNA samples be stored in TE buffer (Tris/EDTA). Tris provides a pH 8.0 environment to keep DNA and RNA nucleases less active. EDTA further inactivates nucleases by binding cations required by nucleases.
Place your tube and those of other groups in a balanced configuration in a microcentrifuge, with cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the silica resin.
Label a clean 1.5-mL tube with your sample number. Transfer 50 μL of the supernatant (clear solution) to the fresh tube. Be careful not to disturb the pellet when transferring the supernatant. Discard old tube containing the silica resin.

Explanation

Transferring silica resin to the PCR reaction in Part III can inhibit the PCR amplification.

Store your sample on ice or at -20° C until you are ready to begin the PCR.

PCR

Goal: Extract DNA from tissue samples

Summary

Steps:

Obtain PCR tube containing Ready-To-Go PCR Bead containing dehydrated Taq polymerase, nucleotides, and buffer. Label the tube with your identification number.
Use a micropipette with a fresh tip to add 23 µL of the appropriate primer/loading dye mix to each tube (refer to primer table below). Allow the beads to dissolve for 1 minute at ambient temperature.
Place the PCR tubes on ice to prevent premature replication of unwanted primer dimers.
Use a micropipette with fresh tip to add 2 µL of your DNA (from Part II) directly into PCR tube with primer and polymerase mixture. Ensure that no DNA remains in the tip after pipetting.

Tip

If the reagents become splattered on the wall of the tube, pool them by briefly spinning the sample in a microcentrifuge (with tube adapters) or by sharply tapping the tube bottom on the lab bench.

If your DNA was extracted using Chelex, allow the tubes containing DNA to sit upright for 10 minutes (or centrifuge for 30 seconds) to ensure that any residual Chelex settles on the bottom of the tubes. When removing DNA for PCR, be careful to only pipet from the very top of the liquid to avoid transferring Chelex into the PCR tube as Chelex inhibits PCR.

Store your sample on ice until your class is ready to begin thermal cycling.
Place your PCR tube, along with those of the other students, in a thermal cycler that has been programmed with the appropriate PCR protocol.
Amplification from some templates, such as the COI barcode region, may be improved by transferring PCR tubes directly from ice into a hot thermal cycler that has been temporarily paused at the beginning of the first 95°C denaturation step. This limits the formation of undesirable primer dimers. Resume the program when all of the PCR tubes are in the thermal cycler.

RbClCOIITS

Primers: (rbcLaF / rbcLa rev)

Temperature	Time	Cyles
94 °C	1:00 minutes	1
94 °C	15 seconds	35
54 °C	15 seconds	35
72 °C	30 seconds	35
4 °C	ad infinitum	1

Primers: (LCO1490 / HC02198)

Temperature	Time	Cyles
94 °C	1:00 minutes	1
94 °C	30 seconds	35
50 °C	30 seconds	35
72 °C	45 seconds	35
4 °C	ad infinitum	1

Primers: (ITS1F / ITS4)

Temperature	Time	Cyles
94 °C	1:00 minutes	1
94 °C	1 minute	35
55 °C	1 seconds	35
72 °C	2 minutes	35
4 °C	ad infinitum	1

AMPure XP Solid Phase Reversible Immobilization (SPRI) Bead PCR Clean-Up (optional)

Summary

This protocol is modified from Beckman Coulter Life Sciences’ protocol for typical PCR cleanup of DNA barcoding PCR products using solid phase reversible immobilization (SPRI) beads (i.e., AMPure XP beads). This protocol will suitably remove the remnants of PCR, selecting for PCR products in the range of 400-800 base pairs, which is consistent with most standard DNA barcode marker regions (e.g., rbcL, COI, etc.). The intent of this protocol is to prepare DNA amplicons for rapid sequencing with ONT’s Rapid Barcoding Kit. Note that it is “optional,” because once all samples are pooled, bead clean-up will still need to be conducted on the pooled amplicon library.

Steps:

Re-suspend AMPure XP beads by using the pipette to mix.
Add the entire PCR reaction (~20 μL) directly to a labeled 1.5 mL microfuge tube containing 36 μL AMPure XP beads. Use the pipette to carefμLly mix the beads and DNA.
Incubate for 5 minutes at room temperature on a microfuge tube rack.
Spin down the tube for 15 seconds and place the tube on a magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid
Keeping the tube on the magnet, pipette off the supernatant and discard without disturbing the pellet.
Keeping the tube on the magnet, add 200 μL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat Step 5.
Remove the tube from the magnetic rack and spin down the tube for 15 seconds. Replace the tube on the magnetic rack and allow the pellet to reform.
Keeping the tube on the magnet, remove any residual liquid (~10-30 μL). With the tube open, allow the pellet to dry for 30 seconds. Caution: Do not allow the pellet to dry to the point of cracking. Be prepared to immediately move on to Step 10.
Remove the tube from the magnetic rack and resuspend the pellet in 10 μL of EB. Use the pipette to continuously wash the EB over the pellet until it completely dissociates.
Incubate the tube for 5 minutes away from the magnetic rack.
Spin down the tube for 15 seconds and replace the tube on the magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid supernatant shoμLd be completely clear.
Remove and retain 10 μL of supernatant. Pipette the supernatant into a thin-walled PCR tube. Discard the tube containing the pellet. The PCR product is now ready for the ONT Rapid Barcode Kit.

Rapid Barcode Index Attachment

Summary

After the optional SPRI bead clean-up step, DNA amplicons are ready for rapid barcode index attachment. Note that up to 24 DNA amplicons can be uniquely tagged during a single library prep and sequencing run using the Rapid Barcode Kit 24 V14. ONT manufactures another kit with up to 96 unique barcode indexes.

Steps

Obtain one unique Rapid Barcode (RB) mix for each PCR amplicon. Record each sample number and its associated RB.
Spin down RB tubes. Pipette 1 μL of each unique RB directly into a PCR tube containing 10 μL of its associated DNA amplicon. Vortex briefly or pipette to mix.
Ensure that PCR tubes are sealed shut, and then incubate tubes in a thermal cycler using the following conditions:

Temperature	Time
30 °C	2:00 minutes
80 °C	2:00 minutes
4 °C	hold

Explanation

The 20 °C incubation step will first ensure optimal transposase activity, promoting the addition of the RB indexes. The 80 °C step will denature and inactivate the transposase to prevent erroneous addition of unique indexes to the wrong DNA amplicons during pooling.

Spin down PCR tubes. DNA amplicons are now uniquely tagged using the ONT RBs.
Pipette each tagged DNA amplicon (~11.5 μL) into a single class-wide 1.5 mL microfuge tube. This represents the pooled amplicon library.

Library Clean-Up with Solid Phase Reversible Immobilization (SPRI) Beads

Summary

Library clean-up will remove remnants of PCR (e.g., primers, polymerase, etc.), residual transposome and unused rapid barcodes, as well as any dyes or indicators used during previous steps (e.g., Cresol red). Note that this step must be carried out even if the optional PCR clean-up step was performed with a class.

Steps:

Re-suspend AMPure XP beads by using the pipette to mix or vortexing.
Add an appropriate volume of AMPure XP beads to the pooled amplicon library using the following formula: VolumeAMPure XP beads= (11.5 μL)(No. of DNA amplicon samples)
Use a pipette to gently mix the beads and amplicon library such that the beads are evenly distributed throughout the tube.
Incubate the mix at room temperature for 10 minutes, flicking occasionally to ensure that beads remain suspended in solution. Note that DNA amplicons are coordinating with SPRI beads during this time.
Spin down the mix for 15 minutes to form an initial bead pellet. Then, place the tube on a magnetic rack to pellet the beads. Allow up to one minute for the beads to pellet. Keeping the tube on the magnetic rack, use an appropriate pipette to remove all of the liquid from the tube and discard. Avoid disturbing the pellet. Remember that the DNA is located in the pellet at this point.
Keeping the tube on the magnetic rack, add 1000 μL of 80% ethanol to the pellet. Then, remove the supernatant and discard without disturbing the pellet.
Repeat Step 6.
Remove the tube from the magnetic rack and spin down the tube for 15 seconds. Replace the tube on the magnetic rack and allow the pellet to reform.
Keeping the tube on the magnet, remove any residual liquid (~10-30 μL). With the tube open, allow the pellet to dry for 30 seconds. Caution: Do not allow the pellet to dry to the point of cracking. Be prepared to immediately move on to Step 10.
Remove the tube from the magnetic rack and resuspend the pellet in 15 μL of EB. Use the pipette to continuously wash the EB over the pellet until it completely dissociates. Note that this may take several repeated washes with the same 15 μL EB.
Incubate the tube for 10 minutes away from the magnetic rack at room temperature.
Spin down the tube for 15 seconds and replace the tube on the magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid supernatant shoμLd be completely clear.
Remove and retain 11 μL of supernatant. Pipette the supernatant into a 1.5 mL microfuge tube. Discard the tube containing the pellet. The pooled amplicon library is now ready for the attachment of rapid adapters and motor proteins.

Rapid Adapter Attachment

Summary

The final step of the library preparation is attaching the motor protein to the ends of the DNA. The Rapid Adapter (RA) reagent contains the motor protein and can attach it to the rapid adaptor chemistry on the ends of the DNA which were added by the transposome complex in a previous step.

Steps:

In a 1.5ml tube dilute Rapid Adapter (RA) as follows:

Reagent	Volume
Rapid Adapter (RA)	1.5ul
Adapter Buffer (ADB)	3.5ul
Total	5ul

Add 1ul of diluted Rapid Adapter to indexed DNA.
Incubate for 10 min at room temperature.

Flow Cell Check

Summary

The flow cell contains the nanopores which facilitate the sequencing. The number of active pores needs to be determined for every flow cell before it is to be used to ensure that there are an adequate number for the sequencing experiment. Because we are sequencing amplicons and don’t need very high-throughput, we suggest using the Flongle flow cell which has a maximum of 126 pores rather than the Minion, which has maximum of 2048.

Steps: 1. Open MinKNOW software. 2. Click the “Start” tab on the upper left of the screen. 3. Click “Flow cell check”

!!! warning "Important' The sequencer will take a few minutes to get to the proper temperature and run the flow cell check. When complete, the number of usable pores on the flow cell will be displayed. The flow cell is considered under warranty if it is checked within 2 weeks of the delivery date, was properly stored at 4 degrees, but has less than 50 pores. If under warranty, contact Oxford Nanopore within 2 days of performing the flow cell check.

Flow Cell Priming

Summary

Before loading the flow cell with the DNA library, the flow cell needs to be “primed” for sequencing. The reagents required for priming and loading the Flongle flow cell are contained in the Flongle Sequencing Expansion (EXP-FSE002), a separate box from the library prep reagents, which contains glass vials of Sequencing Buffer (SB), Flow Cell Flush (FCF), and Library Beads (LIB).

Mix 117ul FCF (from the Flongle expansion kit) and 3ul of FCT (from the Rapid Barcoding kit).
Peel back the seal from the Flongle flow cell.
Slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
Place the pipette tip in the sample port, keeping the pipette vertical.
Turn the dial clockwise to load the flush buffer and primer into the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.

Flow Cell Loading

Make a sequencing mix:

Reagent	Volume
Sequencing Buffer (SB)	15ul
Loading Beads (LIB)	10ul
DNA Library	5ul
Total	30ul

Pipette up sequencing mix, slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
Place the pipette tip in the sample port, keeping the pipette vertical.
Turn the dial clockwise to load to the flush buffer and primer the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.
Place the sticker back on.
Proceed to "Start Sequencing" on MinKNOW software.

Bioinformatics

Full DNA Barcoding Analysis of DNA barcodes can be done using DNA Subway2.0 (coming soon).

Epi2melabs wf-amplicon may also be used to generate consensus sequences for each sample. This conensus sequence can

Data analysis

Goal: Determine the taxa sampled.

Example data

Plant DNA Barcoding :

This dataset was generated at a workshop at James Madison University in June 2024.

Nanopore sequencing report example: Download
FastQ files: Download
Example EPI2ME report: Download

Bioinformatics

This tutorial will use the EPI2ME amplicon workflow developed by Oxford Nanopore.

Workflow version: 1.1.3

Instruction tip

Students can work individually or in groups to analyze their data. Assuming each student sample is assigned their own barcoded samples, they can generate an individual report specific to their example.

Analyze data

Open the EPI2ME software and choose either Sign in or Continue as guest.

Bioinformatics

In this example, we use the Continue as guest option.
Click on the Launch icon and choose the Amplicon workflow.

Bioinformatics

In this example, choose Run Locally.

Failure

If you see the "Setup required" error message, follow the instructions to install or configure any needed software (for example, installing and starting Docker).

Option

Depending on your access and your computer, the launch page is where you will decide on running the workflow locally (on your computer) or on the cloud.
Click on the Launch button to proceed with the workflow.
Under Setup local analysis, complete the required options to load your sequencing dataset and choose analysis parameters.
Bioinformatics

In this example, we use the following parameters:

Input Options
- FASTQ: "fastq_pass" folder provided in this tutorial example data. This folder will be generated by your experiment.
- Analyse unclassified reads: Leave as default.
- Reference: Leave as default.
Sample Options
- All options left as default.
Pre-processing Options
- All other options left as default.
Variant calling options
- All options left as default.
Output Options
- All options left as default.
Advanced Options
- All options left as default.
Miscellaneous Options
- All options left as default.
Nextflow Configuration
- All options left as default.
Nextflow Options
- All options left as default.
Click Launch workflow and then Launch again.

Time

The time required for this analysis depends on your dataset. On a computer with the recommended configuration for running a MinION, the test dataset was completed in approximately 5 minutes.

Interpret results

The wf-amplicon pipeline generates a detailed report (wf-amplicon-report.html) that provides comprehensive insights into sequencing data. However, the file we will need for our analysis in in the folder "output" generated by epi2melabs afer the run. The output folder contains folders for each sample (i.e. "barcode01"), which contain a folder named "consensus". A .fasta file in the consensus folder is a consensus DNA sequence of the DNA Barcoding amplicon from all the sequencing reads for the sample.

The DNA sequence from the .fasta file can be copied and pasted into the blue line of DNA Subway under "Enter sequences in FASTA format".

Instruction tip

DNA Subway 2.0 is coming late Spring/Summer 2025 and is equiped with a new "Blue Line" for Nanopore sequencing data which contains tools for generating the consensus Nanopore sequence reads.

Comments and discussion

See recent comments or start a discussion on our Slack channel.

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Annotated Experiment: 16S Sequencing

Examining Microbial Diversity — Nanopore "Shoe-ome" Sequencing

Protocol information

Protocol Credits

Author(s)
- Jason Williams, Cold Spring Harbor Laboratory
Maintainer/contact: Jason Williams, Cold Spring Harbor Laboratory: email
Last updated: January, 2025
Source materials and references
- Norgen Microbiome Kit
- Swab Collection and DNA Preservation System

DNA sample source

Type: Microbial
Collection source: Collected from swabs of shoes/footwear

Nanopore Sequencing

Sequencing format: Flongle
Sequencing kit: 16S Barcoding Kit 24 V14 (SQK-16S114.24)
Oxford Nanopore Sequencing protocol: Official ONT protocol
Indexed/Barcoded: Yes
Samples per run: 24 samples

Computer and Bioinformatics

Analysis tools
- EPI2ME
  - EPI2ME 16S workflow
Software to download or install:
- Oxford Nanopore Software Downloads Oxford Nanopore account and login required:
  - MinKNOW
  - EPI2ME Desktop Application
- Other software:
  - Docker
Analysis difficulty: Easier
Command line needed: No
GPU/Super-high accuracy basecalling required: No

Reagents

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

Norgen Swab Collection and DNA Preservation System
- Included
  - Sterile swab
  - DNA preservation tube

DNA extraction

Norgen Microbiome DNA Isolation Kit
- Included
  - Lysis Additive A
  - Binding buffer I
  - 70% Ethanol
  - Binding Buffer B
  - Wash Solution A
  - Elution Buffer B
- User-provided
  - 96-100% ethanol
  - 70% ethanol

DNA prep, library creation, and sequencing

Nanopore kit
- Included
  - 16S Barcodes in 96-well plate, at 1 μM each
  - EDTA
  - AMPure XP Beads (AXP)
  - Elution Buffer (EB)
  - Rapid Adapter (RA)
  - Adapter Buffer (ADB)
- User-provided
  - LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  - Nuclease-free water
  - Freshly prepared 80% ethanol in nuclease-free water
  - Qubit dsDNA HS Assay Kit (Invitrogen, Q32851) optional
DNA quality control
- If using Qubit
  - Qubit dsDNA HS Assay Kit (Invitrogen, Q32851) optional
- If using electrophoresis
  - Elecrophoresis grade agarose
  - TBE buffer
  - GelRed/SYBR Green, or other DNA dye for visualization
  - 100bp DNA ladder

Equipment and consumables

Lab equipment

PCR thermocycler
Microcentrifuge (20,000 x g)
Micropipette set (e.g., P10, P100, P1000)
Assorted tube racks (microfuge and PCR tubes)
Magnetic rack for 1.6-1.7ml tubes
Ice bucket with ice
Electrophoresis chamber, power supply, and documentation setup OR Qubit fluorometer
(Optional) Hula mixer
(Optional) Vortexer
Permanent markers

Consumables

Micropipette tips (e.g., P10, P100, P1000)
1.5-1.7ml microfugue tubes
0.2 ml thin-walled PCR tubes
(Optional) 1.5 ml Eppendorf DNA LoBind tubes

Nanopore sequencing equipment

Sequencing device: MinION sequencer (M1kB,C, or D) with Flongel adapter and Flongle flow cells

Computer equipment

Desktop or laptop with MinKNOW and EPI2ME installed

Estimated timings

Collection of microbes from shoe swab: 10 minutes
DNA extraction: 60-75 minutes
PCR, DNA quality control, and library prep:
- PCR: 3-3.5 hours
- Quality control: 10 min (Qubit); 60-75 min (electrophoresis)
- Library prep: 30-60 min
Sequencing: 60 min to 24 hours
Data analysis: 30-60 min

Background

Playlist: 16S Shoe-ome Experiment

Sequencing 16S DNA provides insights into the composition and diversity of microbial communities by targeting the 16S ribosomal RNA (rRNA) gene, (1) a conserved region present in all bacteria. This gene contains variable regions that can differentiate bacterial species and genera, making it a powerful tool for identifying and classifying bacteria in a sample. By analyzing 16S DNA sequences, researchers can determine which bacterial taxa are present, estimate their relative abundances, and compare microbial communities across different environments or conditions. This method is widely used in microbiome studies, environmental monitoring, and pathogen identification.

Read more about 16S Wikipedia

In the "Shoe-ome" experiment, students swab the exterior bottom of their shoes—an easily obtained, personalized sample with minimal risk of accidentally collecting sensitive DNA. High school students with limited or no laboratory experience can complete this experiment in a day-long, 8-hour lab session or across two shorter sessions. DNA is collected from a swab sample, lysed, and concentrated using a commercial kit. The 16S region is amplified, barcoded, and sequenced using the Nanopore kit. The entire 16S rRNA gene is amplified via PCR with barcoded primers, allowing the multiplexing of up to 24 samples in a single sequencing run. The EPI2ME workflow analysis generates several visualizations and characterizations of the diversity and types of microbes detected.

Additional Reading

Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis: paper
Performing accurate species-level bacterial identification with nanopore sequencing: paper
How Bacteria Rule Over Your Body – The Microbiome: video

Collection of microbes from shoe swab

Goal: Obtain a microbial sample for DNA sequencing using the swab kit to sample the bottom of a shoe.

Video: Educator's Guide to Nanopore: Shoe DNA swab

Instruction tip

Each student (up to 24) can prepare their own sample.

Obtain a shoe you wish to sample. The shoe does not need to be visibly dirty; however, a shoe worn daily is likely to have greater microbial diversity than one that has never been worn.
Carefully unscrew the cap of the collection tube without spilling the contents. Next, carefully remove the sterile swab from its package, being cautious not to touch the swab or contaminate it in any way. Dip the swab into the liquid in the collection tube.
Use the moistened swab to gently rub the underside and sides of the shoe. A moist swab is more effective at collecting microbes that may be present on the shoe. Ideally, the swab will visibly change in appearance as it picks up dirt and microbes.
Return the swab to the collection tube. The swab has a "score," a thinned section of the plastic that allows you to break the swab in half, with the half remaining in the tube fitting just enough to be enclosed.
Replace the cap on the collection tube, ensuring that the tube is fully closed and leakproof. The solution in the tube deactivates any microorganisms and preserves DNA. Label the tube with your name and date.

DNA extraction

Goal: Extract and purify DNA from the collected microbes to use in PCR using the microbiome DNA extraction kit.

Video: Educator's Guide to Nanopore: DNA extraction

References

This protocol follows Protocol C from the Norgen Swab Collection and DNA Preservation System.

Part I - Preparing swab sample

Add 100 μL of Lysis Additive A to the swab collection tube and vortex briefly.

Sample bias

The lysis additive and heating steps are meant to break open microbes allowing their DNA to be extracted. Some microbes are tough and may not be efficiently lysed. Keep in mind that difficult-to-lyse or microbes and very rare microbes will be less likely to be detected in your dataset, even if present.
Incubate the swab collection tube at 65˚C for 5 minutes.
Carefully remove the swab from the collection tube.
Label a microcentrifugue tube and transfer up to 1 mL of the preserved sample to the microcentrifuge tube.
Centrifuge the tube for 2 minutes at 20,000 × g (~14,000 RPM). A thin white layer will form on the top of the supernatant.
Label a new microcentrifuge tube and carefully transfer 700 μL of supernatant, without the white layer debris, to the microcentrifuge tube.
Add 100 μL of Binding Buffer I, mix by inverting the tube a few times, and incubate for 10 minutes on ice.
Spin the lysate for 2 minutes at 20,000 x g (~14,000 RPM) to pellet any cell debris.
Label a new microcentrifuge tube. Using a pipette, transfer up to 700 μL of supernatant (avoid contacting the pellet with the pipette tip) into the new microcentrifuge tube.
Add an equal volume of 70% ethanol to the lysate collected above (100 μL of ethanol is added to every 100 μL of lysate). Vortex to mix.

Part II - binding DNA to column

Assemble a spin column with one of the provided collection tubes.
Apply 700 μL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM). Discard the flowthrough and reassemble the spin column with the collection tube.

Tip

Ensure the entire lysate volume has passed through into the collection tube by inspecting the column. If the entire lysate volume has not passed, spin for an additional minute at 20,000 x g (~14,000 RPM).
Repeat step 2 with the remaining volume of lysate mixture.

Part III - wash column

Apply 500 μL of Binding Buffer B to the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM).

Tip

Ensure the entire Binding Buffer B has passed through into the collection tube by inspecting the column. If the entire wash volume has not passed, spin for an additional minute.
Discard the flowthrough and reassemble the spin column with its collection tube.
Apply 500 μL of Wash Solution A to the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM).
Discard the flowthrough and reassemble the spin column with its collection tube.
Repeat steps 3 and 4.
Spin the column for 2 minutes at 20,000 x g (~14,000 RPM) in order to thoroughly dry the resin. Discard the collection tube.

Part IV - Elute DNA

Place the column into a fresh 1.7 mL Elution tube provided with the kit.
Add 50 μL of Elution Buffer B to the column.
Centrifuge for 1 minute at 425 x g (~2,000 RPM), followed by a 1 minute spin at 20,000 x g (~14,000 RPM). If the entire volume has not been eluted, spin the column at 20,000 x g (~14,000 RPM) for 1 additional minute.

Optional

An additional elution may be performed if desired by repeating steps 2 and 3 using 50 μL of Elution Buffer. The total yield can be improved by an additional 20-30% when this second elution is performed.

The purified genomic DNA can be stored at 2-8°C for a few days. For longer term storage, -20°C is recommended.

Pause point

Once DNA extraction is completed you can stop here. However, prepping the PCR can be done in under 20 minutes and since the PCR is long, you may wish to move ahead so that the PCR can run overnight or between lab sessions.

PCR, DNA quality control, and library prep

Goal: Amplify the 16S region from the purified shoe DNA sample and prepare it for sequencing on a flow cell.

Video: Educator's Guide to Nanopore: PCR

Nanopore

This protocol follows the Flongel version of Rapid sequencing DNA - 16S Barcoding Kit 24 V14 (SQK-16S114.24) from Oxford Nanopore.

DNA quantification

The Nanopore protocol requires 10 ng of high molecular weight genomic DNA per barcode. A Qubit fluorometer or Nanodrop spectrophotometer can be used to quantify this small amount of DNA. However, in practice, we often proceed to PCR without quantification, acknowledging the increased risk that some samples may have yields too low to generate a successful PCR product.

Instruction tip

Generally, we recommend the lab instructor prepare and pool the individual samples on the student's behalf. We usually narrate the steps and make use of web cams to allow students to have a close up view.

Take one 96-well plate containing 16S barcodes. Break one set of barcodes (1-24, or as desired) away from the plate and return the rest to storage.
Thaw the desired barcodes at room temperature.
Briefly centrifuge barcodes in a microfuge to make sure the liquid is at the bottom of the tubes and place on ice. If you don't have a plate centrifuge, tap the plate several times to settle the liquid to the bottom.
Thaw the LongAmp Hot Start Taq 2X Master Mix, spin down briefly, mix well by pipetting and place on ice.
For each Shoe DNA sample, add 15μl of sample to a 0.2ml PCR tube. If you have quantified samples, use nuclease-free water to adjust your DNA samples so that your 15μl sample has a total of 10 ng of DNA.

Tip

In a classroom situation, we regularly skip quantification. We also will generally try samples that have a lower-than-optimal concentration of DNA. In general, it is better use err on the side of caution using less DNA rather than overloading with more.
In each 0.2 ml thin-walled PCR tube containing a sample to be tested, prepare the following mixture:

Reagent Volume

10 ng input DNA (from previous step) 15μl

LongAmp Hot Start Taq 2X Master Mix 25μl

Total 40μl
Ensure the components are thoroughly mixed by pipetting and spin down briefly.
Using clean pipette tips, carefully pierce the foil surface of the required barcodes. Use a new tip for each barcode to avoid cross-contamination. Make a note of which barcode numbers will be run for each sample.
Mix the 16S barcodes by pipetting up and down 10 times. Transfer 10 μl of each 16S Barcode into respective sample-containing tubes.
Ensure the components are thoroughly mixed by pipetting the contents of the tubes 10 times and spin down.

Tip

Mix gently to minimize introducing air bubbles to the reactions.

Adding barcodes

Each of the 16S primers in the plate contains one of 24 barcodes. During amplification, PCR products will incorporate a unique barcode sequence and a sequence for the rapid adapter chemistry.

Amplify using the following cycling conditions:

Step	Temperature	Time	Cycles
Initial Denaturation	95 °C	1 min	1
Denature	95 °C	20 sec	3
Anneal	50 °C	30 sec	3
Extend	65 °C	2 min	3
Denaturation	95 °C	20 sec	38
Annealing	55 °C	30 sec	38
Extension	65 °C	2 min	38
Final Extension	65 °C	5 min	1
Hold	4 °C	Infinite	-

Tip

Mix gently to minimize introducing air bubbles to the reactions.

Pause point

With LongAmp Taq, this PCR can take 3 hours or more. Once the PCR reaction is started you can stop here and proceed with library preparation in a subsequent lab session.

Flow cell check

When you are ready to prepare the library (30-45 min), you should proceed directly to loading the flow cells. Now is a good time to check the quality of your flow cell.

Educator's Guide to Nanopore: Library preparation

Thaw reagents at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:

Reagent	Thaw at Room Temperature	Briefly Spin Down	Mix Well by Pipetting or Vortexing
Rapid Adapter (RA)	Not frozen	✓	Pipette
Adapter Buffer (ADB)	✓	✓	Vortex or Pipette
AMPure XP Beads (AXP)	✓	✓	Mix by vortexing immediately before use
Elution Buffer (EB)	✓	✓	Vortex or Pipette
EDTA (EDTA)	✓	✓	Vortex or Pipette

Add 4 µl of EDTA to each barcoded sample, mix thorougly by pipetting and spin down briefly.

Info

EDTA is added at this step to stop the reaction
Incubate for 5 minutes at room temperature.
Check your PCR product

Qubit quantificationElectrophoresis quantification

Follow the dsDNA High Sensitivity protocol to verify the concentration of the product. While you can obtain an absolute quantification, you may still want to examine products via electrophoresis to verify the size of the fragment the the presence of artifacts (e.g primer dimer, spurious products).

You can run PCR products on a 1.5% agarose gel to verify the product and obtain a relative quantification.
Pool all barcoded samples in equimolar ratios in a 1.5 ml tube.

Optional

Eppendorf DNA LoBind tubes are recommended.
Resuspend the AMPure XP Beads (AXP) by vortexing.
To the pool of barcoded samples, add a 0.6X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting:

Volume of Barcoded Sample Pool 37.5μl 75μl 150μl 300μl 600μl

Volume of AMPure XP Beads (AXP) 22.5μl 45μl 90μl 180μl 360μl
Incubate for 5 minutes at room temperature; gently mix by inversion every minute or so; or use a Hula mixer.
Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colorless. Keep the tube on the magnetic rack, and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 1 ml of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.
Remove and retain 15 µl of eluate into a clean 1.5 ml tube (Eppendorf LoBind if using).

Optional

Nanopore recommends here you quantify 1 µl of eluted sample using a Qubit fluorometer. In our experience you may omit this step if you don't have a Qubit.

If you do calculate the concentration, you can meet the loading concentration recommendations (often in femtomoles) using a calculator and assuming a ~1,500bp amplicon.
In a fresh 1.5 ml tube (Eppendorf LoBind if using), dilute the Rapid Adapter (RA) as follows and pipette mix:

Reagent Volume

Rapid Adapter (RA) 1.5μl

Adapter Buffer (ADB) 3.5μl

Total 5μl
Add 0.5µl of diluted Rapid Adapter (RA) to the barcoded DNA.
Mix gently by flicking the tube, and spin down.
Incubate the reaction for 5 minutes at room temperature. Then place the final product on ice while you prepare for loading the flow cell.

Proceed directly to loading

We recommend you proceed to loading your flow cell and sequencing, since the prepared library works best, in our experience, if it is used shortly after prep.

Sequencing

Goal: Load prepared library onto flow cell and sequence.

Video: Educator's Guide to Nanopore: Flongle flow cell loading

Nanopore

This protocol follows the Flongel version of Rapid sequencing DNA - 16S Barcoding Kit 24 V14 (SQK-16S114.24) from Oxford Nanopore.

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
In a fresh 1.5 ml tube (Eppendorf LoBind if using), mix 117µl of Flow Cell Flush (FCF) with 3µl of Flow Cell Tether (FCT) and mix by pipetting.
Place the Flongle adapter into the MinION.
Place the flow cell into the Flongle adapter, and press the flow cell down until you hear a click.
Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed.
To prime your flow cell with the mix of Flow Cell Flush (FCF) and Flow Cell Tether (FCT) that was prepared earlier, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 pipette tip inside the sample port and slowly dispense the 120 µl of priming fluid into the Flongle flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

Tip

The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
Vortex the vial of Library Beads (LIB). Note that the beads settle quickly, so immediately prepare the Sequencing Mix in a fresh 1.5 ml Eppendorf DNA LoBind tube for loading the Flongle, as follows:

Reagents Volume

Sequencing Buffer (SB) 15 µl

Library Beads (LIB) (mixed immediately before use) or Library Solution (LIS) (if using) 10 µl

DNA Library 5 µl

Total 30 µl
To add the Sequencing Mix to the flow cell, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 tip inside the sample port and slowly dispense the Sequencing Mix into the flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.
Seal the Flongle flow cell using the adhesive on the seal tab. Close the MinION and proceed to start the sequencing run.

Nanopore

This protocol follows the Flongel version of Rapid sequencing DNA - 16S Barcoding Kit 24 V14 (SQK-16S114.24) from Oxford Nanopore.

Bioinformatics

For the following steps, you will use the MinKNOW software to operate the MinION device.
Navigate to the start page and click Start sequencing.
Fill in your experiment details, such as name and flow cell position and sample ID.
Select the sequencing kit (16S Barcoding Kit 24 V14 (SQK-16S114.24)) used in the library preparation on the Kit page.
Configure the sequencing parameters for your sequencing run or keep to the default settings on the Run options and Analysis tabs.
Computer with GPUComputer without GPU
We recommend:
- Raw reads: .POD5
- Basedcalled reads: FASTQ
- Basecalling: High-accuracy basecalling (HAC) or Super-accurate basecalling (SUP)
- Modified bases: Off
We recommend:
- Raw reads: .POD5
- Basedcalled reads: FASTQ
- Basecalling: Fast basecalling
- Modified bases: Off
Tip

You may also leave all basecalling off if you will perform that operation on a cloud platform or more powerful computer.
On the Output page, set up the output parameters or keep to the default settings.
Click Start on the Review page to start the sequencing run.

Pause point

In most cases, you can generate useful data to analyze within an hour. A more complete dataset will take about 24 hours (the usual lifetime of a Flongle).

Data analysis

Goal: Determine the microbes present in the shoe sample and their taxonomic relationships.

Example data

Guam Shoe-ome:

This dataset was generated at a workshop at JFK High School, March 2024.

Nanopore sequencing report example: Download
FastQ files: Download
Example EPI2ME report: Download

Bioinformatics

This tutorial will use the EPI2ME 16s workflow developed by Oxford Nanopore.

Workflow version: 1.1.3

Instruction tip

Students can work individually or in groups to analyze their data. Assuming each student sample is assigned their own barcoded samples, they can generate an individual report specific to their example.

Analyze data

Open the EPI2ME software and choose either Sign in or Continue as guest.

Bioinformatics

In this example, we use the Continue as guest option.
Click on the Launch icon and choose the 16S workflow.

Bioinformatics

In this example, choose Run Locally.

Failure

If you see the "Setup required" error message, follow the instructions to install or configure any needed software (for example, installing and starting Docker).

Option

Depending on your access and your computer, the launch page is where you will decide on running the workflow locally (on your computer) or on the cloud.
Click on the Launch button to proceed with the workflow.
Under Setup local analysis, complete the required options to load your sequencing dataset and choose analysis parameters.
Bioinformatics

In this example, we use the following parameters:

Input Options
- FASTQ: "fastq_pass" folder provided in this tutorial example data. This folder will be generated by your experiment.
- Bam: Leave as default (blank).
- Classification method: Leave as default (minimap2).
- Analyze unclassified reads: Leave as default (unchecked).
- Exclude host reads: Leave as default (blank).
Real-Time Analysis Options
- All options left as default.
Tip

You can start your EPI2ME analysis while sequencing is ongoing. This allows you to show a real-time analysis of your data rather than waiting until sequencing is completed.

Sample Options
- All options left as default.
Reference Options
- Choose a database: "SILVA_138_1".
- All other options left as default.
Kraken2 Options
- All options left as default.
Minimap2 Options
- Compute coverage and sequencing depth of the references: Checked.
- All options left as default.
Report Options
- All options left as default.
Advanced Options
- All options left as default.
Miscellaneous Options
- All options left as default.
Nextflow Options
- All options left as default.
Click Launch workflow and then Launch again.

Time

The time required for this analysis depends on your dataset. On a computer with the recommended configuration for running a MinION, the test dataset was completed in approximately 35 minutes.

Interpret results

The wf-16s pipeline generates a detailed report (wf-16s-report.html) that provides comprehensive insights into sequencing data and microbial community composition. Below are the major components of the report:

Read Statistics Summary

This section presents an overview of the sequencing reads, including:
- Total read counts
- Quality metrics
- Length distributions
These metrics help assess the overall quality of the sequencing run.
Taxonomic Composition

Displays the identified taxa within the sample, often visualized through:
- Bar plots or pie charts illustrating the relative abundances of different microbial groups.
Diversity Metrics

Includes calculations of:
- Alpha diversity indices, such as Shannon and Simpson indices, which assess diversity within a sample.
- Beta diversity analysis for comparing differences between multiple samples (if applicable).
Reference Alignment Statistics

For workflows using Minimap2 for taxonomic classification, this section provides:
- Alignment statistics, including sequencing depth and coverage for each reference genome.
- Heatmaps visualizing sequencing depth across genomic coordinates for the most covered references.
Rarefaction Curves

Plots display the mean species richness as a function of sampling effort, helping to determine if the sequencing depth was sufficient to capture the sample's diversity. Learn about rarefaction curves.
Abundance Tables

Provides detailed tables listing the counts of different taxa per sample.
- Customizable filters (e.g., abundance_threshold) can exclude low-abundance taxa for clearer analysis.
These components collectively offer a robust analysis of sequencing data, enabling a deeper understanding of microbial communities and their diversity.

Instruction tip

In depth explanations of the report metrics are available on the 16S workflow github documentation page.

Bioinformatics

Things to look for

There are a variety of results contained in the 16S report. While it is not possible to describe all outcomes there are several things to draw your attention to.

Read Quality
- Uncorrected Nanopore reads have a lower quality score, especially with fast basecalling. You can expect uncorrected reads in the 8-12 phred score range. Using super accurate basecalling is one way you can improve this result.
Read Length
- A sharp peak around 1500bp—the length of the 16S amplicon—should be expected.
Samples summary
- Ideally, you will have an about equal number of reads from all barcodes. This is difficult to achieve, and barcodes with lower reads will be under-sampled relative to more abundant samples. You could prepare a new library to adjust if something critical was underrepresented.
- You will occasionally get a few (<10) reads from barcodes that were not used in your experiment. Sequencing error may misidentify a barcode, misclassifying it. This is normal.

Comments and discussion

See recent comments or start a discussion on our Slack channel.

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Annotated Experiment: Phage Sequencing

Sequencing Phage Genomes with Oxford Nanopore Technologies (ONT)

Summary: Students can isolate and sequence high-quality bacteriophage genomes.

Acknowledgements

We would like to acknowledge the SEA-PHAGES team, Ximena Iraheta, Michael Shamash and Corinne Maurice for their help in isolating the phages used as examples. We would also like to thank the Biology Department at Spelman College, especially Maira Goytia and Shrijeeta Ganguly, and funding source NSF through ‘Developing Foundations for Nanopore DNA Sequencing Course-based Undergraduate Research Experiences at Minority-Serving Institutions’ (Award 2215496).

Protocol information

Protocol Credits

Authors
- James T. Melton III, Spelman College, Atlanta, GA, USA
- Kaitlyn Parrish, Spelman College, Atlanta, GA, USA
- Jordan Dotson, Spelman College, Atlanta, GA, USA
- Brenna Fox, McGill University, Montreal, Qc, Canada
- Hui-Min Chung, University of West Florida, Pensacola, FL, USA
- Patrick Lypaczewski, McGill University, Montreal, Qc, Canada
Maintainer/contact: James T. Melton III, Spelman College: email
Last updated: March, 2025
Source protocols and references
- Promega Wizard® DNA Clean-Up System - A7280
- SEA-PHAGES 9.1 DNA extraction
- (alternative) Norgen Phage DNA isolation Kit - 46800
- (alternative) Sera-Xtracta Virus/Pathogen Kit
- (alternative) Pathogen/Viral Nucleic Acid Isolation Kit (Magnetic Bead System)

DNA source material

Type: Viral
Collection source: Phage isolates

Nanopore Sequencing

Sequencing format: MinION
Sequencing kit: One of the following kits (See protocol for recommendations based on your endpoints)
Oxford Nanopore Sequencing protocol:
Indexed/Barcoded: Yes, 24-96 depending on kit
Samples per run: 24-96 of samples

Computer and Bioinformatics

Analysis tools
- Google Colab:
  - Dorado basecaller
  - Flye assembler
- Galaxy:
  - Nanoplot
  - Flye assembler
Analysis difficulty: Challenging
Command line needed: Yes
GPU/Super-high accuracy basecalling required: Yes (Provided by the online bioinformatics platforms)

Choosing kits and formats

Depending on how many samples and students you want to process, you can either choose rapid barcoding or native barcoding with either 24 or 96 indicies. An explanation of the options are below.

Kits	Rapid Barcoding Sequencing Kit 24 V14 (SQK-RBK114.24) or Rapid Barcoding Sequencing Kit 96 V14 (SQK-RBK114.96)	Native Barcoding Kit 24 V14 (SQK-NBD114.24) or Native Barcoding Kit 96 V14 (SQK-NBD114.96)
Number of genomes	Up to 24 or 96 samples can be multiplexed, depending on the chosen kit
3rd party reagents for reaction	None	Yes (Enzymes from NEB or Roche)
Required DNA	• 50 ng per sample for > 4 samples (5 ng/µl) • 200 ng per barcode if ≤ 4 samples (20 ng/µl)	400 ng per sample for > 4 samples (≥36.3 ng/µl) 1000 ng per barcode if ≤ 4 samples (≥90.9 ng/µl) NOTE: 11-12 µl of a DNA sample are used during library prep. The concentrations above are ideal for sequencing; however, samples as low as 5.8 ng/µl (63.8 ng) have been sequenced successfully, even when pooled with samples prepared with the recommended amounts of DNA.
Application	• Entry-level CURE projects, such as identifying cluster type of isolated phages (similar but more advanced to the DOGEMS strategy in SEA-PHAGES or Science Education Alliance- Phage Hunters Advancing Genomics and Evolutionary Science) • Advanced level of CURE projects, such as phage genome annotation and genomic comparisons • Exploring phage diversity from environmental samples.
Pros	• Can be performed in class due to a shorter library prep time (estimated: 50 minutes). • No third-party reagents are required (less expensive).	• Will allow for sequencing of the terminal ends of the phage genomes. • Long fragment buffer (LFB) allows for size selection of DNA fragments > 3000 bp. NOTE: While using the long fragment buffer might decrease the concentration of DNA, genome assembly works best with longer reads.
Cons	• Due to the transposase-based library preparation, the terminal ends of the phage genomes might not be sequenced. • Genomic DNA is cut randomly, which can result in shorter read sizes if the genomic extraction wasn’t done carefully.	• Due to the lengthy process (estimated: 10-12 hours, depending on the number of samples), it is recommended that the instructor perform the library prep outside of class time; however, there are pausing points in the protocol. • Third-party reagents are required (more expensive).

Reagents

Info

In this protocol, we will detail a phage DNA extraction, sequencing, and genome assembly, using the recommended phage isolation and DNA extraction protocols by the SEA-PHAGES (Science Education Alliance- Phage Hunters Advancing Genomics and Evolutionary Sciences) program.

Info

We organized the options in the following DNA extraction protocols into Easiest option (EO) or Lower cost (LC). For example, most labs are more familiar with spin column-based DNA extraction kits, but magnetic bead kits allow for smaller aliquoting volumes and lower costs. Similarly, purchasing magnetic racks from scientific vendors such as New England Biolabs (NEB) or ThermoFisher is generally more accessible for universities with strict purchasing policies than purchasing 3rd party supplies or 3D printing your own racks, although it can offer substantial cost savings. Additionally, we have focused our library preparation protocol on the Native Barcoding Kit 24 V14; however, the rapid barcoding kits have also been used to successfully sequence phage DNA.

Easiest Option (EO)Lowest Cost (LC)

References

This list assumes you are using the Promega Wizard® DNA Clean-Up System - A7280 for DNA extraction.

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

See Phage isolation and culturing instructions from SEA-PHAGES

DNA extraction

High titer phage lysates (≥ 9 x 10⁵ PFU/mL or plaque forming units per milliliter)

Tip

Medium titer phage lysates (10⁷ - 10⁹ PFU/mL) may provide enough DNA for sequencing.
Promega Wizard® DNA Clean-Up System - A7280

Tip

Several silica column kits exist from any number of competitors: Norgen Phage DNA Isolation kit or others.
- Included
  - 2 ml DNA clean-up resin (Promega Wizard DNA Clean-Up Kit)
  - 2 DNA clean-up columns (Promega Wizard DNA Clean-Up Kit)
- User-provided. Optional reagents were used here
  - Nuclease mix - preparation instructions:
    - DNase 1
    - RNase A
    - NaCl
    - Glycerol
    - ddH2O
  - 80 % isopropanol, freshly prepared
  - 3 ml syringes
  - Proteinase K (20 mg/ml) and SDS (10%) (optional)
  - 0.5 M EDTA (optional)
  - ddH2O pre-warmed (95 °C)

DNA prep, library creation, and sequencing

Nanopore kit
- Native Barcoding Kit 24 V14.
  - Native Adapter (NA)
  - Sequencing Buffer (SB)
  - Library Beads (LIB) OR Library solution (LS)
  - Elution buffer (EB)
  - AMPure XP beads (AXP)
  - Long Fragment Buffer (LFB) OR Short Fragment Buffer (SFB)
  - EDTA
  - Flow Cell Flush (FCF)
  - Flow Cell Tether (FCT)
  - Native Barcode Plate
User-provided reagents
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Nuclease-free water (e.g., ThermoFisher, AM9937
- Freshly prepared 80% ethanol in nuclease-free water
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g, Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

References

This list assumes you are using the Sera-Xtracta Virus/Pathogen Kit for DNA extraction.

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

See Phage isolation and culturing instructions from SEA-PHAGES

DNA extraction

High titer phage lysates (≥ 9 x 10⁵ PFU/mL or plaque forming units per milliliter)

Tip

Medium titer phage lysates (10⁷ - 10⁹ PFU/mL) may provide enough DNA for sequencing.
Sera-Xtracta Virus/Pathogen Kit
- Included
  - Binding/Lysis Reagent
  - Wash Buffer
  - SeraSil-Mag 400 beads
  - SeraSil-Mag 700 beads
  - Proteinase K liquid
- User-provided
  - Ethanol (absolute)
  - Nuclease free water

DNA prep, library creation, and sequencing

Nanopore kit
- Native Barcoding Kit 24 V14.
  - Native Adapter
  - Sequencing Buffer
  - Library Beads OR Library solution
  - Elution buffer
  - AMPure XP beads
  - Long Fragment Buffer OR Short Fragment Buffer
  - EDTA
  - Flow Cell Flush
  - Flow Cell Tether
  - Native Barcode Plate
User-provided reagents
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Nuclease-free water (e.g., ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g, Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Equipment and consumables

Easiest Option (EO)Lowest Cost (LC)

References

This list assumes you are using the Promega Wizard® DNA Clean-Up System - A7280 for DNA extraction.

Lab equipment

Micropipette set (e.g., P2, P10, P100, P200, P1000) and tips
Microcentrifuge (20,000 x g)
Heat block or water bath
Hula mixer (gentle rotator mixer)
Qubit (dsDNA HS Assay Kit - ThermoFisher, Q32851; and assay tubes) or Nanodrop spectrophotometer for DNA quantification
Permanent markers
Magnetic rack for microfuge tubes
PCR machine (for incubation of samples in PCR tubes)
Microplate centrifuge, e.g., Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
Vortex mixer
Ice bucket
Timer

Consumables

Assorted tube racks (microfuge, conical tubes, and PCR tubes)
Sterile 15 ml conical tubes
1.5-1.7 ml microfugue tubes
0.2 ml thin-walled PCR tubes
(Optional) 1.5 ml and 2 ml Eppendorf DNA LoBind tubes
(Optional) Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat #0030129504) with heat seals

Nanopore sequencing equipment

Sequencing device: MinION

Computer equipment

Desktop or laptop for MinION device; internet access for online bioinformatics tools

References

This list assumes you are using the Sera-Xtracta Virus/Pathogen Kit for DNA extraction.

Lab equipment

Micropipette set (e.g., P2, P10, P100, P200, P1000) and tips
Microcentrifuge (20,000 x g)
Heat block or water bath
Hula mixer (gentle rotator mixer)
Qubit (dsDNA HS Assay Kit - ThermoFisher, Q32851; and assay tubes) or Nanodrop spectrophotometer for DNA quantification
Permanent markers
Magnetic rack for microfuge tubes
PCR machine (for incubation of samples in PCR tubes)
Vortex mixer
Ice bucket
Timer
(Optional) Microplate centrifuge, e.g., Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)

Consumables

Micropipette set (e.g., P10, P100, P1000) and tips
(Optional) pipette tips with aerosol barrier
Assorted tube racks (microfuge, conical tubes, and PCR tubes)
Sterile 15ml conical tubes
1.5-1.7ml microfuge tubes
(Optional) 1.5 ml and 2 ml Eppendorf DNA LoBind tubes
(Optional) Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat #0030129504) with heat seals

Nanopore sequencing equipment

Sequencing device: MinION

Computer equipment

Desktop or laptop for MinION device; internet access for online bioinformatics tools

Estimated timings

Extraction and quantification of phage genomic DNA

Promega Wizard® DNA Clean-Up System - A7280: 1 hour for an instructor to extract one sample; 1.5-2 hours for a class of 10 students
Qubit: 10 to 30 min depending on number of samples

Library preparation for sequencing

Rapid barcoding kit: 50 min
Native barcoding kit: 10-12 hours for an instructor depending on the number of samples; if needed, there are pausing points

Sequencing

1-3 days depending on the number of samples

Base calling and reads analysis

For a whole sequencing run, a few hours to a few days depending on the number of reads, the basecalling model (i.e., fast, high accuracy, or super accurate), and system specifications.

Background

Bacteriophages (phages) are viruses that infect and replicate inside a bacterial host. Phages are the most abundant biological entities on Earth (estimated 10³¹), and their vast diversity remains largely unexplored even after decades of research. Whole genome sequencing (1) of isolated phages can determine the precise number and sequence of nucleotides that make up an entire genomic DNA (gDNA) molecule. This data allows researchers to explore phage diversity with a complete set of phage genes (e.g., DNA packaging, structural, DNA replication, lysis, lysogeny, anti-host defense genes, auxiliary metabolic genes), group phages into clusters, and perform genome comparisons.

Read more about whole genome sequencing Wikipedia

In this phage genome experiment, students extract double-stranded gDNA from high titer lysates (≥ 9 x 10⁵ PFU/mL), quantify DNA concentrations, prepare DNA libraries for Nanopore sequencing, prime and load a MinION flow cell, and perform data analysis (i.e., basecalling, quality control, and genome assembly). Up to 96 phage isolates can be multiplexed on a single MinION flow cell, which allows every student to continue to take ownership of their project by sequencing their own isolate in the classroom. This research experience is designed to teach undergraduate students during a two-semester program, Phage Discovery and Phage Genomics, as implemented by most SEA-PHAGES Institutions; however, it is possible that a condensed version could be completed in one semester.

Additional Reading and Information

Cresawn, S. G., Bogel, M., Day, N., Jacobs-Sera, D., Hendrix, R. W., & Hatfull, G. F. (2011). Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC bioinformatics, 12, 1-15. https://doi.org/10.1186/1471-2105-12-395

De Coster, W., D’hert, S., Schultz, D. T., Cruts, M., & Van Broeckhoven, C. (2018). NanoPack: visualizing and processing long-read sequencing data. Bioinformatics, 34(15), 2666-2669. https://doi.org/10.1093/bioinformatics/bty149

Kieft, K., Zhou, Z., & Anantharaman, K. (2020). VIBRANT: automated recovery, annotation and curation of microbial viruses, and evaluation of viral community function from genomic sequences. Microbiome, 8, 1-23. https://doi.org/10.1186/s40168-020-00867-0

Kolmogorov, M., Yuan, J., Lin, Y., & Pevzner, P. A. (2019). Assembly of long, error-prone reads using repeat graphs. Nature Biotechnology, 37(5), 540-546. https://doi.org/10.1038/s41587-019-0072-8

Moraru, C. (2023). VirClust—A tool for hierarchical clustering, core protein detection and annotation of (prokaryotic) viruses. Viruses, 15(4), 1007. https://doi.org/10.3390/v15041007

Moraru, C., Varsani, A., & Kropinski, A. M. (2020). VIRIDIC—A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses, 12(11), 1268. https://doi.org/10.3390/v12111268

Nishimura, Y., Yoshida, T., Kuronishi, M., Uehara, H., Ogata, H., & Goto, S. (2017). ViPTree: the viral proteomic tree server. Bioinformatics, 33(15), 2379-2380. https://doi.org/10.1093/bioinformatics/btx157

Oxford Nanopore Technologies. Dorado. https://github.com/nanoporetech/dorado

Rinehart, C. A., Gaffney, B., Wood, J. D., & Smith, J. P. (2016). a Phage Evidence Collection and Annotation Network. https://discover.kbrinsgd.org/login

Russell DA, Hatfull GF. (2017). PhagesDB: the actinobacteriophage database. Bioinformatics, 33(5): 784-786. https://doi.org/10.1093/bioinformatics/btw711

SEA-PHAGES Phage Discovery Guide. https://seaphagesphagediscoveryguide.helpdocsonline.com/home

SEA-PHAGES Phage Genomics Guide. https://genomicsguide.seaphages.org/

The Galaxy Community. (2024). The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update, Nucleic Acids Research https://doi.org/10.1093/nar/gkae410

Wang, R. H., Yang, S., Liu, Z., Zhang, Y., Wang, X., Xu, Z., Wang, J., & Li, S. C. (2024). PhageScope: a well-annotated bacteriophage database with automatic analyses and visualizations. Nucleic Acids Research, 52(D1), D756-D761. https://doi.org/10.1093/nar/gkad979

Wick, R. (2017). Filtlong. In GitHub repository. GitHub. https://github.com/rrwick/Filtlong

Extraction and quantification of phage genomic DNA

Easiest Option (EO)Lowest Cost (LC)

Goal: Extract genomic phage DNA from high-titer lysate.(1)

Video: Phage Discovery Protocol: DNA Extraction

References

This DNA extraction protocol uses the Promega Wizard® DNA Clean-Up System - A7280 and is taken from Protocol 9.1 in the SEA-PHAGES Discovery Guide

Supplies and reagent preparation for DNA extraction (1 sample)

Included in kit
- 2 ml DNA clean-up resin. Note: Thoroughly mix the Wizard® DNA Clean-Up Resin before taking an aliquot. If crystals or aggregates are present, warm the resin to 37°C for 10 minutes to dissolve them. The resin itself remains is insoluble. Allow it to cool to 25–30°C before use.
- 2 DNA clean-up columns
User-provided. Note: Optional reagents were used for the DNA extraction of phages BillyTP and Bhageatrice used as example data.
- 1 ml phage lysate (titer ≥ 5 x 10⁹ PFU/ml)
- 5 µl Nuclease mix
- 12 ml 80 % isopropanol, freshly prepared
- 3 ml syringes
- 0.5 µl Proteinase K (20 mg/mL) and 50 µl SDS (10%) (optional)
- 15 µl 0.5 M EDTA (optional)
- ddH2O pre-warmed (95 °C)

Degrade bacterial DNA/RNA in high-titer phage lysate

Aseptically transfer 1 ml of phage lysate into a microcentrifuge tube.
Wearing gloves and working in the designated area, add 5 µl nuclease mix to the lysate.

Important

The enzymes (RNase in particular) are very stable and can persist and contaminate equipment and supplies throughout the laboratory. Take precautions to keep and use them in the designated area.
Mix gently but thoroughly by repeated inversions—do not vortex!
Incubate at 37 °C for 10 minutes or room temperature for 30 minutes.
Remove and discard your gloves before returning to your bench.

Optional

[6]. Add 15 µl EDTA to the nuclease-treated lysate and mix gently. EDTA will inactivate the nucleases by chelating, or binding, divalent cations required by the nucleases for activity.

[7]. Add 0.5 µl Proteinase K and 50 µl SDS to the nuclease-treated lysate and mix gently. Incubate at 37 °C for 10 minutes. Proteinase K is added to degrade the nucleases added in Step 2. SDS stimulates the activity of Proteinase K.

Denature the protein capsid to release phage DNA

Put on a fresh pair of gloves.
Add 2 ml of DNA clean-up resin to a 15 ml conical tube.
1. The DNA resin is a slurry solution containing microscopic polymer beads. Make sure that the bottle of resin is well mixed, the precipitate dissolved by heating to 37 °C, and the beads resuspended before aliquoting your 2 ml. (Your instructor may have done this for you.)
Warning

The resin contains guanidinium thiocyanate, a chemical that denatures proteins. Do not get it on your skin!
Transfer your nuclease-treated phage lysate from the microcentrifuge tube to the 15 ml conical tube containing resin.
Mix the solution by gently inverting the tube repeatedly for 2 minutes.

Isolate the phage genomic DNA

Label two Wizard Kit columns with your initials.
Remove the plungers from two 3 ml syringes and attach a column to each syringe barrel.
Follow the steps below for each column at the same time:
1. Set the column and syringe barrel on a new microcentrifuge tube.
2. Transfer 1.5 ml of phage DNA/resin solution to the column using a pipette.
3. Do not discard the empty 15 ml conical tube.
4. Insert a plunger into the syringe and carefully push all the liquid through, collecting the flow-through in the used 15 ml conical tube from above.
  
  Important
  
  The DNA is bound to the polymer beads that pack into the column as the liquid is pushed through. It is VERY important to maintain a firm, gentle, unrelenting, and even pressure on the syringe. Do not let the plunger pop out of the syringe barrel because releasing the vacuum will ruin the column.
5. Once the liquid is expelled, maintain pressure on the plunger as you dry residual liquid by touching the tip of the column to a paper towel.
6. Unscrew the column from the syringe barrel before releasing the plunger and set the column into a clean microcentrifuge tube.
7. Remove the plunger from the syringe barrel, and then reattach the syringe barrel to the column.
Wash the salts from the DNA (now in the column) with the following steps for each column:
1. Add 2 ml 80 % isopropanol to each syringe barrel/column and push the liquid through the column, repeating steps 3(d)–3(f).
2. Repeat twice, for a total of three isopropanol washes.
Remove residual isopropanol.
1. With each column in a fresh 1.5 ml microcentrifuge tube, spin at 10,000 × g for 5 minutes.
  1. The column will prevent the microfuge tube lids from closing. Arrange the open tubes in the centrifuge so that the lids point toward the center of the rotor.
2. Transfer columns to new 1.5 ml microcentrifuge tubes. Spin at 10,000 × g for 1 additional minute to remove any residual isopropanol.
3. Evaporate the last traces of isopropanol by removing your columns from the microcentrifuge tubes and placing them directly in a 90 °C heating block for 60 seconds.
  
  Important
  
  Leaving the columns in the heat block for more than 1 minute can lead to DNA damage.
Elute the phage DNA from the columns.
1. Place each column in a clean microcentrifuge tube and apply 50 μl of 90 °C sterile ddH2O directly to each column.
  
  Important
  
  Keep the ddH2O in the heating block so that it remains at 90 °C.
2. Incubate columns for 1 minute at room temperature.
3. Spin at 10,000 × g for 1 minute in a microcentrifuge.
4. Combine the products from both microcentrifuge tubes into one tube; this is your eluted phage DNA.

Determine the concentration of your phage DNA

Using a spectrophotometer (fluorimeter, or Nanodrop) and a protocol from your instructor, quantify your phage DNA.
Place at 4 °C for short-term storage (1–2 weeks) or at -20 °C for long-term storage.

Goal: Extract genomic phage DNA from high-titer lysate.

Alternatives to Promega Wizard

Other kits, such as the Pathogen/Viral Nucleic Acid Isolation Kit (Magnetic Bead System) from Norgen, are also likely to work.
A fully customized extraction kit is also possible, using third-party sourced or homemade reagents such as Lysis buffer (e.g., 2% SDS, 100 mM Tris-HCl ph8.0, 50 mM EDTA), Proteinase K solution (e.g., QIAGEN), and magnetic beads (e.g., Cytiva Sera Mag) to further reduce per sample costs. However, due to the large sizes in which individual reagents are sold and optimization, a fully customized extraction kit may require large upfront costs.

References

This DNA extraction protocol is taken from Sera-Xtracta Virus/Pathogen Kit

Reagent preparation before use of kit

80% ethanol wash solution (Wash 2)

Prepare an 80% ethanol wash solution. Note: Prepare enough 80% Ethanol for 950 μL per extraction reaction. Use 100% absolute ethanol and nuclease-free water.

SeraSil-Mag bead working solution

Prepare a working solution of SeraSil-Mag 400 and SeraSil-Mag 700 beads (supplied as separate vials in the kit) in a 1:1 ratio. Vortex SeraSil-Mag beads thoroughly before each is added to the pre-mixture and then again prior to use. Note: Prepare sufficient bead volume for 20 μL bead mixture per reaction.

Lysis and nucleic acid binding

Add 10 μL of Proteinase K Solution to a 1.5–2.0 mL microcentrifuge tube.
Add 100–400 μL of sample to the tube.

Tip

The Sera-Xtracta Virus/Pathogen Kit from Cytiva was tested down to ¼ volumes (384 total extractions per kit).
Add 20 μL of SeraSil-Mag bead working solution (as prepared above) and mix the solution by slowly pipetting up/down 5–10 times.

Note

Prior to adding, ensure SeraSil-Mag bead tubes are thoroughly vortexed and mix the beads frequently during pipetting.
Add 570 μL of binding/lysis reagent to the 1.5–2.0 mL microcentrifuge tube.

Note

Solution is highly viscous; pipette slowly to avoid a void volume in the tip and excess foaming.
Ensure thorough mixing of the solution, cap and place tube on vortex mixer set a medium speed for 1 minute. Pulse spin contents in a microcentrifuge to bring contents down.
Incubate tube on heat block set to 60°C for 10 minutes.

Note

Heat step enhances lysis and activates Proteinase K enzyme. If precipitation is evident, quick spin tube to bring down contents.
Place tube on magnet stand for 1 minute or until the solution becomes clear. Without disturbing bound beads, carefully remove the entire supernatant.

Wash the Bound RNA/DNA

Remove the sample tube from the magnet stand and add 950 μL Wash Buffer (Wash 1) to the sample tube. Mix by slowly pipetting the contents 5–10 times.
Ensure thorough mixing of the solution, cap and place tube on vortex mixer set a medium speed for 1 minute. Pulse spin contents in a microcentrifuge to bring contents down.
Place the tube on a magnet stand for 1 minute or until the solution becomes clear. Without disturbing bound beads, carefully remove the entire supernatant.
Remove the tube from the magnet stand and add 950 μL of freshly prepared 80% ethanol (Wash 2) to the sample tube.
Mix tube contents by slowly pipetting the contents up/down 5–10 times. Pulse spin contents in a microcentrifuge to bring contents down.
Place the tube on the magnet stand for 1 minute or until the solution becomes clear. While on the magnet stand, carefully remove the supernatant without disturbing the pellet.
Briefly remove the tube from the magnet stand allowing the beads to sink towards the bottom of the tube. Note: This should take 3–5 seconds.
Place the tube back onto the magnet. When the beads collect to the magnet use a 10 or 20 μL pipette to carefully remove any remaining ethanol.

Important

It is important to ensure that all the ethanol is removed.
Remove tube from magnetic stand and allow beads to dry for 2 minutes.

Elution of RNA/DNA

Add 50 μL of pre-heated (70°C–75°C) nuclease-free water to each sample tube. Pipette up and down slowly until all the beads are removed from the side of the tube and the entire bead mass is at the bottom of the tube (pulse spin the tubes in a microcentrifuge if needed).
Place the tube on the magnet stand for 1 minute or until contents are clear.
With the tube on the magnet, carefully transfer the eluate, containing the extracted RNA/DNA sample to a new microcentrifuge tube.

Note

A pulse spin in a microfuge is strongly recommended before magnet settling to ensure all the liquid sample in the tube is collected together in a single bulk volume at the bottom of the tube. Isolated droplets on the tube walls or trapped under the tube lid will affect results.

Storage of recovered nucleic acid

The protocol recommends elution of the sample in nuclease free water. Purified DNA or RNA maybe stored at 2°C–8°C for a short period when used immediately for analysis and/or downstream molecular biology/analytical applications. For long term storage aliquot and store purified DNA isolates at -20°C and RNA isolates at -80°C or less, the user might consider using standard TE buffer, pH 8–8.5 for DNA or 1 mM sodium citrate, pH 6.5 for RNA.

Quantification of DNA

Goal: Determine DNA concentration for library preparation.

Accurate DNA quantification is essential to normalize DNA inputs during library prep. Too much DNA from one sample may overwhelm the flow cell, resulting in fewer reads for other samples. An Invitrogen™ Qubit™ 4 Fluorometer is recommended for the most accurate DNA concentration readings.

References

This protocol is taken from General Qubit Assay Protocol

Set up two assay tubes for the standards and one assay tube for phage each sample.
Prepare the Qubit™ working solution by diluting the Qubit™ reagent 1:200 in Qubit™ buffer. Prepare 200 μL of working solution for each standard and sample.

Prepare the assay tubes according to the table

Tube	Standards	Phage sample
Working solution* (from step 2)	190 μL	180-199 μL
Standard (from kit)	10 μL	—
User sample	—	1–20 μL
Total Volume in each assay tube	200 μL	200 μL

*Qubit 1X dsDNA assays (Cat. Nos. Q33230, Q33231, Q33265, Q33266) are supplied with a ready-to-use working solution, and do not require preparation.

Vortex all tubes for 2–3 seconds.
Incubate the tubes for 2 minutes at room temperature.
Insert the tubes in the Qubit™ Fluorometer and take readings. For detailed instructions, refer to the Qubit™ Fluorometer manual.

Tip

Qubit gives very accurate quantification of DNA. However, Nanodrop quantification gives absorbance, allowing you to determine the quality and "cleanness" of your extraction. Pure DNA A260/A280 ratio is about 1.8.

Info

Since we will follow the library preparation for the native barcoding kit, you will ideally need 400 ng of gDNA per sample if using >4 barcodes OR 1000 ng gDNA per sample if using ≤4 barcodes; however, phage genomes with as little as 63.8 ng have successfully been sequenced and assembled.

Flow cell check

When you are ready to prepare the library, you should proceed directly to loading the flow cells. Now is a good time to check the quality of your flow cell.

Library Preparation - Native Barcoding Prep (24)

Goal: Attach sequencing barcodes and adaptors necessary for Nanopore sequencing.

Nanopore

This protocol follows the MinION version of Native Barcoding Kit 24 V14 from Oxford Nanopore.

Materials, consumables, and equipment

Materials
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- 400 ng gDNA per sample if using >4 barcodes
- OR 1000 ng gDNA per sample if using ≤4 barcodes
Consumables
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml and 2 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g., ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g, Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- (Optional) Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat #0030129504) with heat seals
Equipment
- Hula mixer (gentle rotator mixer)
- Microfuge
- Magnetic rack
- Vortex mixer
- Thermal cycler
- P1000, P200, P100, P20, P10, P2 pipettes and tips; Optional: Multichannel pipette and tips
- Eppendorf 5424 centrifuge (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Microplate centrifuge, e.g., Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Ice bucket with ice
- Timer

DNA repair and end-prep

Goal: Ensure DNA ends are enzymatically prepared to attach barcodes.

Thaw the AMPure XP Beads (AXP) and DNA Control Sample (DCS) at room temperature and mix by vortexing. Keep the beads at room temperature and store the DNA Control Sample (DCS), if using, on ice.
Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
NEB enzyme prep instructions
1. Thaw all reagents on ice.
2. Flick and/or invert the reagent tubes to ensure they are well mixed.
  
  Note
  
  Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
3. Always spin down tubes before opening for the first time each day.
4. The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
  
  Note
  
  It is important the buffers are mixed well by vortexing.
5. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
Important
- Do not vortex the NEBNext FFPE DNA Repair Mix or NEBNext Ultra II End Prep Enzyme Mix.
- It is important that the NEBNext FFPE DNA Repair Buffer and NEBNext Ultra II End Prep Reaction Buffer are mixed well by vortexing.
Dilute your DNA Control Sample (DCS) by adding 105 µl Elution Buffer (EB) directly to one DCS tube. Mix gently by pipetting and spin down.

Tip

Oxford Nanopore recommends using the DNA Control Sample (DCS) in your library prep for troubleshooting purposes. However, you can omit this step and make up the extra 1 µl with your sample DNA.
In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), prepare your DNA samples:
- For >4 barcodes, aliquot 400 ng per sample
- For ≤4 barcodes, aliquot 1000 ng per sample
Make up each sample to 11 µl using nuclease-free water. Mix gently by pipetting and spin down.

Combine the following components per tube/well; between each addition, pipette mix 10 - 20 times:

Reagent	Volume
DNA sample	11 µl
Diluted DNA Control Sample (DCS)	1 µl
NEBNext FFPE DNA Repair Buffer	0.875 µl
Ultra II End-prep Reaction Buffer	0.875 µl
Ultra II End-prep Enzyme Mix	0.75 µl
NEBNext FFPE DNA Repair Mix	0.5 µl
Total	15 µl

Tip

Oxford Nanopore recommends making up a mastermix of the end-prep and DNA repair reagents for the total number of samples and adding 3 µl to each well.

Ensure the components are thoroughly mixed by pipetting and spin down in a centrifuge.
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
Resuspend the AMPure XP beads (AXP) by vortexing.
Add 15 µl of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard. If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repeat the previous step.
Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Dispose of the pelleted beads.
Quantify 1 µl of each eluted sample using a Qubit fluorometer.

Pause point

Take forward an equimolar mass of each sample to be barcoded forward into the native barcode ligation step. However, you may store the samples at 4°C overnight.

Native barcode ligation

Goal: Ligate barcodes to genomic DNA samples.

Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:
1. Thaw the reagents at room temperature.
2. Spin down the reagent tubes for 5 seconds.
3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Thaw the EDTA at room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the Native Barcodes (NB01-24) at room temperature. Briefly spin down, individually mix the barcodes required for your number of samples by pipetting, and place them on ice.
Select a unique barcode for each sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment. Only use one barcode per sample.
In clean 0.2 ml PCR-tubes or a 96-well plate, add the reagents in the following order per well; Between each addition, pipette mix 10 - 20 times.

Reagent Volume

End-prepped DNA 7.5 µl

Native Barcode (NB01-24) 2.5 µl

Blunt/TA Ligase Master Mix 10 µl

Total 20 µl
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate for 20 minutes at room temperature.
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly. Note: Ensure you follow the instructions for the cap colour of your EDTA tube. EDTA is added at this step to stop the reaction.

EDTA Cap Colour Volume per Well

Clear Cap EDTA 2 µl

Blue Cap EDTA 4 µl

Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube. Note: Ensure you follow the instructions for the cap colour of your EDTA tube.

	Volume per Sample	For 6 Samples	For 12 Samples	For 24 Samples
Total volume for preps using clear cap EDTA	22 µl	132 µl	264 µl	528 µl
Total volume for preps using blue cap EDTA	24 µl	144 µl	288 µl	576 µl

Tip

Oxford Nanopore recommends checking the base of your tubes/plate are all the same volume before pooling and after to ensure all the liquid has been taken forward.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add 0.4X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting. Note: Ensure you follow the instructions for the cap colour of your EDTA tube.

	Volume per Sample	For 6 Samples	For 12 Samples	For 24 Samples
Volume of AXP for preps using clear cap EDTA	9 µl	53 µl	106 µl	211 µl
Volume of AXP for preps using blue cap EDTA	10 µl	58 µl	115 µl	230 µl

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
Keep the tube on the magnetic rack and wash the beads with 700 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard. If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repeat the previous step.
Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 35 µl nuclease-free water by gently flicking.
Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µl of each eluted sample using a Qubit fluorometer.

Pause point

Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, you may store the sample at 4°C overnight.

Adapter ligation and clean-up

Goal: Ligate sequencing adapters to barcoded genomic DNA.

Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:
1. Thaw the reagents at room temperature.
2. Spin down the reagent tubes for 5 seconds.
3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes. Note: Do NOT vortex the Quick T4 DNA Ligase.
Note

The NEBNext Quick Ligation Reaction Buffer (5x) may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.

Important

Do not vortex the Quick T4 DNA Ligase.
Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
Important

Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally.
- To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB); We recommend using this buffer.
- To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)
Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and keep at room temperature.
In a 1.5 ml Eppendorf LoBind tube, mix in the following order; Between each addition, pipette mix 10 - 20 times:

Reagent Volume

Pooled barcoded sample 30 µl

Native Adapter (NA) 5 µl

NEBNext Quick Ligation Reaction Buffer (5X) 10 µl

Quick T4 DNA Ligase 5 µl

Total 50 µl
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 20 minutes at room temperature.

Important

The next clean-up step uses Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) rather than 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 20 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
Wash the beads by adding either 125 μl Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
Remove the tube from the magnetic rack and resuspend pellet in 15 µl Elution Buffer (EB).
Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube; Dispose of the pelleted beads
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Depending on your DNA library fragment size, prepare your final library in 12 µl of Elution Buffer (EB).

Fragment Library Length Flow Cell Loading Amount

Very short (<1 kb) 100 fmol

Short (1-10 kb) 35–50 fmol

Long (>10 kb) 300 ng
Note
- If the library yields are below the input recommendations, load the entire library.
- If required, we recommend using a mass to mol calculator such as the NEB calculator.

End of library prep

The prepared library is used for loading onto the flow cell. Store the library on ice or at 4°C until ready to load.

Tip

Library storage recommendations

Oxford Nanopore recommends storing libraries in Eppendorf DNA LoBind tubes at 4°C for short-term storage or repeated use, for example, re-loading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes.

If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells. Depending on how many flow cells the library will be split across, more Elution Buffer (EB) than what is supplied in the kit will be required.

Priming and loading the flow cell

Goal: Load prepared library for sequencing.(1)

Video and guide: Priming and loading your flow cell

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.

Tip

BSA: For optimal sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), Oxford Nanopore recommends adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml.

Loading Beads or Solution: For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).
To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.
Note

Oxford Nanopore is in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
- Single-use tubes format: Add 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml and 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).
- Bottle format: In a suitable tube for the number of flow cells, combine the following reagents:
  
  Reagent Volume per Flow Cell
  
  Flow Cell Flush (FCF) 1,170 µl
  
  Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl
  
  Flow Cell Tether (FCT) 30 µl
  
  Total Volume 1,205 µl
Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the priming port cover to ensure correct thermal and electrical contact.

Flow cell check

Complete a flow cell check to assess the number of pores available before loading the library.This step can be omitted if the flow cell has been checked previously. See the flow cell check instructions in the MinKNOW protocol for more information.
Slide the flow cell priming port cover clockwise to open the priming port.

Important

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 200 µl
2. Insert the tip into the priming port
3. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip. Note: Visually check that there is continuous buffer from the priming port across the sensor array.
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.

Important

The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:

Reagent	Volume per Flow Cell
Sequencing Buffer (SB)	37.5 µl
Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using	25.5 µl
DNA Library	12 µl
Total	75 µl

Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
Place the light shield onto the flow cell, as follows:
1. Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.
2. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.
Close the device lid and set up a sequencing run on MinKNOW.

Sequencing on MinKNOW

Phage Sequencing Tips

Sequencer	Description
MinION Mk1B (recommended)	This sequencer uses MinION flow cells and requires an external computer for acquisition and base-calling. (IT requirements)
MinION Mk1C (discontinued)	This sequencer used MinION flow cells in an integrated mini-computer to provide an ‘all-included’ sequencing experience. It has been discontinued and is being replaced by the MinION Mk1D iPad accessory.
MinION Mk1D	This sequencer uses MinION flow cells and requires an external computer for acquisition and base-calling. It replaces the Mk1B with better temperature control and USB-C connectivity. (IT requirements)
Flongle	This adapter converts any MinION flow cell-capable sequencer to use Flongle flow cells. (MinION Mk1B IT requirements)
GridION	This sequencer is a desktop computer with five integrated MinION flow cell slots. (IT requirements)
PromethION	This sequencer is a large production-scale machine better suited for larger projects than bacteriophage genomes. (IT requirements)

Flow Cells (Pores / Hours)	Details
Flongle	>50 pores / 16 hours
MinION	>800 pores / 72 hours
PromethION	>5000 pores / 72 hours

Sequencing Time Sequencing time varies per flow cell and multiplexing.

Flongle flow cells are generally used and disposed of after their entire lifespan.
MinION or PromethION flow cells can be used in short bursts (1-6h) and either continued or reset using a wash kit and reused with other samples if sufficient data has been captured. Unwashed flow cells can be stored at 4℃ until analysis is completed to continue acquisition if data is insufficient.

Basecalling	Description
File Formats	Nanopore sequencing produces two primary file formats: FASTQ and POD5. The raw data generated by nanopore electrical trace signals are stored in POD5 files, which are then converted to FASTQ files through basecalling. FASTQ files contain nucleotide sequences and quality scores. FASTQ files can always be regenerated from POD5 files using different models.
Fast or High Accuracy (HAC)	Sufficient for phage cluster identification and initial assessment of sequencing depth (bases sequenced / ~50 kB average phage genome size).
Super Accurate (SUP)	Highest quality available, reaching 98-99.5% raw read accuracy. Reduces the chances of assembly errors and is recommended for publication.
Notes / Recommendations	Basecalling can be performed with Dorado, integrated within MinKNOW, either during sequencing or after. It can also be run as a standalone tool. SUP basecalling is computationally intensive. If the minimum computer requirements are not met, we recommend performing basecalling on Google Colab or a supercomputing cluster.

Bioinformatics

For the following steps, you will use the MinKNOW software to operate the MinION device.

Navigate to the start page and click Start sequencing.
Fill in your experiment details, such as name and flow cell position and sample ID.
Select the sequencing kit (Native Barcoding Kit 24 V14 (SQK-NBD114.24)) used in the library preparation on the Kit page.
Configure the sequencing parameters for your sequencing run or keep to the default settings on the Run options and Analysis tabs.
Computer with GPUComputer without GPU
We recommend:
- Raw reads: .POD5
- Basecalled reads: FASTQ
- Basecalling: High-accuracy basecalling (HAC) or Super-accurate basecalling (SUP)
- Modified bases: Off
We recommend:
- Raw reads: .POD5
- Basecalled reads: FASTQ
- Basecalling: Fast basecalling
- Modified bases: Off
Tip

You may also leave all basecalling off if you will perform that operation on a cloud platform or more powerful computer.
On the Output page, set up the output parameters or keep to the default settings.
Click Start on the Review page to start the sequencing run.

Pause point

In most cases, you can generate useful data to analyze within an hour. A more complete dataset will take about 24 hours.

Data analysis - basecalling, quality control, and assembly

Goal: Use cloud platforms to basecall data, perform quality control, and assemble phage genomes.

Example data

Example Nanopore data can be downloaded from the 'Example phage data' Google Drive folder, and assemblies can be compared to the Illumina assemblies on the Actinobacteriophage Database at phagesdb.org.

Cluster AY Arthrobacter globiformis phage BillyTP
• Basecalling: File BillyTP_0.pod5 contains the raw signal data. Due to the size of POD5 files, only one pod5 file with a subset of the data is provided for the basecalling tutorial and does not contain all of the reads used to assemble the genome below.
• Quality control and Assembly: Three fastq files (BillyTP_0.fastq, BillyTP_1.fastq, BillyTP_2.fastq) contain the ‘sup@v5.0’ basecalled reads needed for assembly. When compared with the PhagesDB reference that was sequenced with Illumina (GenBank Accession Number: PP978841), the contig assembled with Flye (53,003 bp; ~710X) varied by one single nucleotide base at position 46,969, which is likely a “growth” mutation and not due to a sequencing or assembly error. NOTE: A smaller contig is also assembled, which is the ~3-4 kb positive control phage Lambda.

Cluster AY Arthrobacter globiformis phage Bhageatrice
• Quality control and Assembly: Three fastq.gz files (Bhageatrice_0.fastq.gz, Bhageatrice_1.fastq.gz, and Bhageatrice_2.fastq.gz) contain the ‘sup@v5.0’ basecalled reads needed to assemble a 54,699 bp contig (~954X coverage). The Nanopore assembly is 100% identical to the Illumina assembly provided on the PhagesDB phage page.

Cluster FE Arthrobacter globiformis phage CabbageMan
• Basecalling: File cabbageman.pod5 contains the raw signal data for ~347X the CabbageMan genome needed to baseball into a FASTQ file.
• Quality control and Assembly: File cabbageman.v5.fastq contains the ‘sup@v5.0’ basecalled reads needed to assemble the genome using Flye and produces the exact same 15,612bp assembly as the PhagesDB reference that was sequenced with Illumina (GenBank Accession Number: PQ362679).

Bioinformatics

For the following steps, you will use the Dorado software on Google Colab to perform basecalling.

Basecalling with Dorado using Google Colab (convert POD5 to FASTQ)

Before genome assembly, basecalling must be performed to convert POD5 files to FASTQ files. See sample POD5 files of phages BillyTP and CabbageMan.

Basecalling with Dorado using Google Colab (convert pod5 to fastq)

Go to Google Colab. Purchase compute units (if needed). Click on the solid down arrow (upper right), click ‘View resources’, click ‘Learn more’ to purchase computer units.
Click on ‘Connect’ (upper right) and select ‘T4 GPU’ under ‘Hardware accelerator’. NOTE: L4 and A100 GPUs will perform faster but cost more to run. Phage genomes are generally small and do not require heavy computational resources.
Add code by clicking ‘+ Code’. Connect notebook to Google Drive storage; click play symbol to run code (~20 s). NOTE: Text can be added to the notebook by clicking ‘+ Text’.
```
from google.colab import drive
drive.mount('/content/drive')
```
Upload pod5 data. Alternatively, the files can be used from Google Drive.
Download the Dorado software (1 min). Please see https://github.com/nanoporetech/dorado to select the appropriate path as versions and instructions may change in the future. (linux-x64 is needed for Google Colab)
```
!wget https://cdn.oxfordnanoportal.com/software/analysis/dorado-0.7.1-linux-x64.tar.gz
```
Decompress/unpack the Dorado software (1 min).
```
!tar -xvf dorado-0.7.1-linux-x64.tar.gz
```
Run super accurate (SUP) basecalling with Dorado to convert the POD5 files into FASTQ files (~3-4 min). Models can be specified as ‘fast’, ‘hac’, ‘sup’ for the latest model available or explicitly as ‘sup@v5’.
```
!dorado-0.7.1-linux-x64/bin/dorado basecaller --emit-fastq sup BillyTP_barcode11_0.pod5 >BillyTP_barcode11_0.fastq
```
NOTE: The above command will work when using a single phage or pre-demultiplexed POD5 such as the example data provided here. When using a native or rapid barcoding kit, the below two step commands are needed.

7b.
```
    !dorado-0.7.1-linux-x64/bin/dorado basecaller  --kit-name SQK-RBK114-24 sup@v5 multiplexdata.pod5 > intermediatefile.bam

    !dorado-0.7.1-linux-x64/bin/dorado demux --emit-fastq --no-classify --output-dir demultiplexed-fastqs intermediatefile.bam
```
Optional: Gzip the FASTQ file (~ 5 s).
```
!gzip BillyTP_barcode11_0.fastq
```
Copy ‘.fastq’ or ‘.fastq.gz’ file to your Google Drive or download it to your computer. Files will not save in Google Colab.
```
!cp BillyTP_barcode11_0.fastq.gz /path/to/directory
```

Bioinformatics

For the following steps, you will use the Nanoplot software on Galaxy for quality control of reads.

Read summary statistics using Nanoplot on Galaxy

This is a tutorial for summary statistics of the read number, length, and quality using fastq or fastq.gz files. Use sample fastq or fastq.gz data of phages BillyTP, Bhageatrice, and CabbageMan.

Read summary statistics using Nanoplot on Galaxy

Go to Galaxy; if needed, create a free account. All accounts come with a 250 Gb disk quota.
Add a new history by clicking the ‘+’ on the upper right hand side of the screen and name the history. Click ‘Save’.
Upload data (FASTQ or FASTQ.GZ) on the upper left hand side of the screen. Drag and drop file(s) to the screen or select ‘Choose local file’. Click start to upload the file(s). On the right hand side of the screen, files that have been uploaded successfully will turn green on the right hand side of the screen.

Upper left hand side of screen

Middle of screen
Search for and click on ‘NanoPlot’ tool
Select FASTQ or FASTQ.GZ file if using one file and click ‘Run Tool’. You can also change the option under ‘Select multifile mode’ from ‘batch’ to ‘combined’ to add multiple files.

View ‘HTML report’ file on screen by clicking the eye icon; if needed, download figures from ‘HTML report’ and summary statistics from ‘NanoStats’. Summary statistics and plots can be found below.

Filtering reads (optional): Short (e.g., <5 kb) and low quality reads (e.g., <10 Q-score) can be filtered out and visualized with Nanoplot. If needed, Filtlong on Galaxy can be used to filter out reads to make a new filtered fastq file. Alternatively, Nanofilt can be installed to filter fastq files. Depending on the dataset, more stringent filtering options can be set. Filtering was not required to assemble the high quality genomes from the sample data.

Cluster AY Phage	BillyTP (Fall 2023)	Bhageatrice (Fall 2024)
fragment buffer used during library prep*	Short fragment buffer (SFB)	Long fragment buffer (LFB)
Reads contain positive control phage Lambda (3-4 kb)*	yes	no
Genome size (bp)*	53,003	54,699
number of reads	12,121	4,674
number of bases	41,633,273	53,595,600
median read length	906	8,232.5
mean read length	3,434.8	11,466.8
read length stdev	7,515.7	12,187.2
N50	12,870	21,560
mean qual	13.8	18.4
median qual	15.4	20.8
Reads >Q10:	11,602 (95.7%) 40.2Mb	4,673 (100.0%) 53.6Mb
Reads >Q15:	6,828 (56.3%) 28.7Mb	4,307 (92.1%) 49.9Mb
Reads >Q20:	225 (1.9%) 0.2Mb	2,848 (60.9%) 34.5Mb
Reads >Q25:	2 (0.0%) 0.0Mb	251 (5.4%) 0.3Mb
Reads >Q30:	0 (0.0%) 0.0Mb	86 (1.8%) 0.0Mb

Nanoplot summary statistics of the sequence data used to assemble cluster AY phages BillyTP (Genome: 53,003 bp) and Bhageatrice (Genome: 54,699 bp). There are two main reasons why the mean read lengths are shorter for BillyTP: 1) the fastq files contain a short fragment of the positive control phage Lamda (3-4 kb); 2) the short fragment buffer (SFB) was used during library prep. The long fragment buffer allowed for size selection of DNA fragments that were >3,000 bp, and therefore, longer read lengths were sequenced in phage Bhageatrice. The longer, higher quality reads of phage Bhageatrice reduced the total number of reads needed for genome assembly. *Information not provided by Nanoplot.

Figure 1 (A-D). Nanoplot plots of the read lengths (A, B) and read lengths vs. average read quality (C, D) for phages BillyTP (A, C) and Bhageatrice (B, D). The blue arrows in Figs. 2A and B are showing reads that are approximately the size of the phage genomes.

Genome assembly, annotation, and analysis tools

Category	Description & Resources
Assembly	Resources for assembly Flye (Kolmogorov et al. 2019) • Galaxy • Google Colab • EPI2ME (desktop/cloud): The bacterial genome assembly workflow can be used for assembly until a virus-specific workflow is available. Computer requirements for EPI2ME desktop: • Recommended: CPUs = 16; Memory = 64GB • Minimum: CPUs = 8; Memory = 32GB Notes & recommendations: Aim for 100-1000X coverage. Coverage that is too low or too high can cause errors in the assembly; however, successful assemblies have been obtained from as low as 20X, provided the reads were of high quality and length. Post-assembly editing of the genome termini might be required. Google Colab Pro Pricing: Google Colab Pro costs $9.99/month for 100 compute units. Alternatively, a ‘Pay as you go’ option allows purchasing 100 compute units for $9.99. Compute units expire after 90 days. This should be sufficient to run approximately 25 to 50 assemblies of medium-sized phage genomes (~40-60 kbp). Additional resources: BV-BRC
Annotation	Resources for manual annotation: • SEA-PHAGES Phage Genomics Guide • The Actinobacteriophage Database at phagesdb.org • PECAAN (Rinehart et al. 2016; User Manual) Resources for automatic annotation: • VIBRANT (Kieft et al. 2020) • PhageScope (Wang et al. 2024) • Phagenomics Note: Automatic annotations should be double-checked before genomes are finalized.
Analysis	Resources for genomic analysis and comparisons: • Phamerator (Cresawn et al. 2011): Run locally by installing from GitHub or visualize genomes on the Phamerator website • VIRIDIC (Moraru et al. 2020): Run locally by installing from GitHub or run on VIRIDIC website • VirClust (Moraru 2023): Run locally by installing from Github or run on VirClust website • ViPTree (Nishimura et al. 2017): Run locally by installing from Github or run on the ViPTree server

Bioinformatics

For the following steps, you will use the Flye software on Galaxy to assemble reads.

Genome Assembly with Flye on Galaxy

The number of reads needed to assemble a phage genome will vary depending on the size and complexity of the genome and the length and quality of the reads. Most phage genomes should be able to assemble with Flye on Galaxy; however, if more RAM is needed, the assembly can be run on Google Colab (see following tutorial) or a supercomputing cluster. See sample FASTQ or FASTQ.GZ data of phages BillyTP, Bhageatrice, and CabbageMan.

Genome assembly with Flye on Galaxy

Go to Galaxy; if needed, create an account. All accounts come with a 250 Gb disk quota.
Add data to a previous history or click the ‘+’ (upper right) to create a new history. Click the pencil icon (Edit) to rename history.
Click ‘Upload’ (upper left) and drag and drop or select the ‘fastq’ or ‘.fastq.gz’ files from your computer. Click ‘Start’ to upload your files. Files that have been uploaded will turn green on the right hand side of the page.

Upper left hand side of screen

Middle of screen

Right hand side of screen
Go to ‘search tools’ and search for and click on ‘Flye’
Select the ‘fastq.gz’ files that you would like to assemble, and run with default options:
1. Mode: Nanopore raw (--nano-raw). NOTE: Nanopore HQ (--nano-hq) can also be selected if the reads are high quality
2. Number of polishing iterations (1)
3. Keep haplotypes (no)
4. Enable scaffolding using graph (no)
5. Perform metagenomic assembly (no)
6. Reduced contig assembly coverage (disable reduced coverage for initial disjointing assembly). Can enable if needed.
7. Remove all non-primary contigs from the assembly (no)
8. Generate a log file (no). Can select ‘yes’ if needed.
9. Additional options: email notification. Select ‘yes’, if needed.
Click ‘Run Tool’
Outputs. The run has started when the outputs on the right hand side have turned tan and have finished when they have turned green. The following are the outputs. The eye icon (Display) can be used to view the file. Each output can be downloaded by clicking on the output name and selecting the floppy disk icon (Download).
1. Consensus: a fasta file containing the contigs
2. Assembly graph
3. Graphical fragment assembly
4. Assembly info: length of contig(s), coverage (aim for 100-1000X), circular (yes or no), repeat (yes or no)

Bioinformatics

For the following steps, you will use the Flye software on Google Colab to assemble reads.

Genome Assembly with Flye using Google Colab

If more RAM is needed to run a genome assembly, Google Colab is a good option. The number of reads needed for a quality assembly of a phage genome will vary depending on the size of the genome and the length and quality of the reads. See sample fastq or fastq.gz data of phages BillyTP, Bhageatrice, and CabbageMan.

Genome assembly with Flye using Google Colab

Go to Google Colab.
Purchase compute units (if needed). Click on the down arrow (upper right), click ‘View resources’, and then click ‘Learn more’ to purchase more units.
Click on ‘Connect’ (upper right) and select ‘T4 GPU’ under ‘Hardware accelerator.’
Add code or text by clicking ‘+ Code’ or ‘+ Text’, respectively. Connect notebook to Google Drive storage (optional); click play symbol to run code (~20 s).
```
from google.colab import drive  
drive.mount('/content/drive')
```
Upload ‘fastq.gz’ files. On left hand side of screen, click on the folder icon (Files) and then click on ‘Upload to session storage’ icon. NOTE: This data will not be saved and any outputs need to be saved if outputs are not directed to Google Drive.
Download Flye from github. Please see https://github.com/mikolmogorov/Flye to select the appropriate path as versions and instructions may change in the future. (linux-x64 is needed for Google Colab)
```
!wget https://github.com/fenderglass/Flye/archive/refs/tags/2.9.tar.gz
```
Unzip file.
```
!tar -xzf /content/2.9.tar.gz
```
Change directory to Flye
```
!cd Flye-2.9/
```

Build ‘setup.py’

!python3 /content/Flye-2.9/setup.py build

Install ‘setup.py’

!python3 /content/Flye-2.9/setup.py install

Run Flye. NOTE: If you uploaded your Google Drive, you can select the ‘fastq.gz’ files from your folder and direct the outputs to a Google Drive folder. You can also add your .fastq.gz files one by one to the command instead of running all by *.fastq.gz. If the files are not gzipped, change the code to *.fastq.
```
!flye --nano-hq *.fastq.gz --out-dir /content/sample_data --threads 4 --iterations 1
```
Important outputs to save. NOTE: Files will be deleted after the runtime stops.
1. On screen: Total length, Fragments, Fragments N50, Largest fragment, Scaffolds, and Mean Coverage (aim for 100-1000X)
2. In folder:
  • assembly.fasta: contains contigs in fasta format
  • assembly_info.txt: contains contig number, length (bp), mean coverage , circular (yes/no), repeat (yes/no)
Optional: Other options to run
```
!flye --help
```
Phage Contig size (bp) Coverage circular (Y/N)

BillyTP 53,003 710X yes

Bhageatrice 54,699 954X yes

Genome assembly summary information for phages BillyTP and Bhageatrice. The Nanopore assembly of BillyTP only varied by one base pair when compared to the Illumina assembly (GenBank Accession number: PP978841.1). This variation is likely due to a “growth” mutation and not sequencing or assembly error. The Nanopore assembly of Bhageatrice was 100% identical to the Illumina assembly that is available on the Actinobacteriophage Database at PhagesDB.org.

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Annotated Experiment: Microbial Whole Genome Sequencing

Sequencing whole genomes of microbial isolates

Summary: In this experiment, students used microbiology techniques to culture and isolate bacteria from soil collected in their backyards. Starting with those cultures, we sequence whole bacterial genomes.

Protocol information

Protocol Credits

Author(s)
- Jason Williams, Cold Spring Harbor Laboratory
Maintainer/contact: Jason Williams, Cold Spring Harbor Laboratory: email
Last updated: March, 2025
Source materials and references
- NEB Monarch HMW DNA Tissue Kit
  - See also: Protocol Guidance for Extraction of Ultra-High Molecular Weight (UHMW) Genomic DNA for Ultra-Long (UL) Read NGS Sequencing applications in Oxford Nanopore Technologies® workflows
- Qubit™ dsDNA Quantification Assay - High Sensitivity

DNA sample source

Type: Microbial
Collection source: Bacterial isolates cultured on LB Agar from soil.

Nanopore Sequencing

Sequencing format: MinION
Sequencing kit: Native Barcoding Kit 24 V14 (SQK-NBD114.24)
Oxford Nanopore Sequencing protocol: Official ONT protocol
Indexed/Barcoded: Yes
Samples per run: 24 samples

Computer and Bioinformatics

Analysis tools
- EPI2ME
- Jupyter Notebook
Software to download or install:
- Oxford Nanopore Software Downloads Oxford Nanopore account and login required:
  - MinKNOW
  - EPI2ME Desktop Application
- Other software:
  - Docker
Analysis difficulty: More difficult
Command line needed: Some
GPU/Super-high accuracy basecalling required: Yes, if possible.

Reagents

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

Collection and isolation of bacteria not covered in this protocol. You may fine these two articles helpful
Bacterial Isolation
Soil Macromorphology: Isolation of Soil Bacteria

DNA extraction

NEB Monarch HMW DNA Tissue Kit
- Included
  - Monarch HMW gDNA Tissue Lysis Buffer
  - Monarch Protein Separation Solution
  - Monarch gDNA Wash Buffer
  - Monarch RNase A (store -20°C after opening)
  - Proteinase K, molecular biology grade (store -20°C after opening)
  - Elution buffer plus; see Protocol Guidance for Extraction of Ultra-High Molecular Weight (UHMW) Genomic DNA for Ultra-Long (UL) Read NGS Sequencing applications in Oxford Nanopore Technologies® workflows ]
- User-provided
  - Phosphate buffered saline (PBS), cold
  - Ethanol (≥ 95%)
  - Isopropanol
  - For Gram-negative bacteria: Lysozyme (25 mg/ml)
  - For Gram-positive bacteria: STET Buffer containing Lysozyme (10 mg/ml); potentially also lysostaphin
  - Triton-X

DNA quantification

Qubit™ dsDNA Quantification Assay - High Sensitivity
- Included
  - Qubit™ dsDNA HS buffer
  - Qubit™ dsDNA HS standard #1
  - Qubit™ dsDNA HS standard #2

DNA prep, library creation, and sequencing

Nanopore kit
- Included
  - DNA Control Sample (DCS)
  - Native Adapter (NA)
  - Sequencing Buffer (SB)
  - Elution Buffer (EB)
  - AMPure XP Beads (AXP)
  - Long Fragment Buffer (LFB)
  - EDTA (EDTA)
  - Flow Cell Flush (FCF)
  - Flow Cell Tether (FCT)
  - Native Barcode plate (NB01-24)
- User-provided
  - NEB Blunt/TA Ligase Master Mix (NEB, M0367)
  - NEBNext FFPE Repair Mix (NEB, M6630)
  - NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
  - NEBNext Quick Ligation Module (NEB, E6056)
  - Nuclease-free water (e.g. ThermoFisher, AM9937)
  - Freshly prepared 80% ethanol in nuclease-free water
  - Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Equipment and consumables

Lab equipment

PCR thermocycler
Microcentrifuge (20,000 x g)
Micropipette set (e.g., P10, P100, P1000)
Assorted tube racks (microfuge and PCR tubes)
Magnetic rack for 1.6-1.7ml tubes
Ice bucket with ice
Qubit™ fluorometer
Permanent markers
(Optional) Thermal mixer containing a 1.5 ml tube block
(Optional) Vertical rotating mixer/Hula mixer
(Optional) Rotor-stator homogenizer
(Optional) Vortexer
(Optional) Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)

Consumables

NEB Monarch HMW DNA Tissue Kit
- Included
  - Monarch DNA capture beads
  - Monarch bead retainers
  - Monarch 2 ml tubes
  - Monarch spin collection tubes
Qubit™ dsDNA Quantification Assay - High Sensitivity
- Included
  - Qubit™ assay tubes
Micropipette tips (e.g., P10, P100, P1000)
1.5-1.7ml microfugue tubes
0.2 ml thin-walled PCR tubes
(Optional) wide-bore 1000ul pipette tips
(Optional) 1ml syringes and 25G blunt needles
(Optional) 1.5 ml Eppendorf DNA LoBind tubes
(Optional) Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) for working with 24 or more samples.

Nanopore sequencing equipment

Sequencing device: MinION sequencer (M1kB,C, or D)

Computer equipment

Desktop or laptop with MinKNOW and EPI2ME installed

Estimated timings

DNA extraction: 90-120 minutes + elution time (1-24 hours)
DNA quality control, and library prep:
- Quality control: 10 min (Qubit); 60-75 min (if using electrophoresis)
- Library prep: 90-120 min
Sequencing: 24-72 hours
Data analysis: Varies (a few hours)

Microbiology Safety Protocols

We recommend reviewing ASM Guidelines for Biosafety in Teaching Laboratories when working with bacterial cultures.

Background

Playlist: Nanopore Sequencing: Microbial Isolates

Sequencing microbial isolates offered students a hands-on opportunity to explore the rich biodiversity of bacteria in their own environment—and potentially uncover organisms with important applications. In fact, many novel antibiotics, including those effective against drug-resistant pathogens, have been discovered by sequencing bacteria isolated from soil samples (Terramycin was the blockbuster antibiotic that Pfizer discovered in soil samples). At the 2024 DNA Learning Center Sequence-a-Genome camp, high school students participated in an exciting experiment involving the sequencing of bacterial samples collected from their own backyard soil samples. Students extracted DNA from each isolate and generated long-read sequence data to identify, characterize, and compare the microbes they discovered. This project connected cutting-edge genomics to real-world challenges in medicine and biotechnology, all starting from the ground beneath their feet.

Additional Reading

Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing: paper
Sequencing and analysis of nanopore-only microbial isolates with the NO-MISS workflow: video
Isolation and Screening of Soil Bacteria as a Source of Novel Antibiotics: video

DNA Isolation (Part I) — Lysis and precipitation

Goal: Lyse bacterial cells and precipitate high molecular weight DNA.

Video: Educator's Guide to Nanopore—Microbial Isolates: DNA Isolation I (Lysis and Precipitation)

Instruction tip

Each student (up to 24) can prepare their own sample. If handling bacterial samples is undesirable, it may be possible to prepare lysates to the precipitation step. Appropriate safety protocols should still be observed.

References

This DNA extraction protocol follows Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Bacteria (NEB #T3060) and Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow; both from New England Biolabs.

NEB has published the following overview of the Monarch® HMW DNA Extraction Kit workflow; keep in mind the timing they suggest will likely be much different in a classroom setting.

Bacterial lysis

Cell count and Gram classification

Cell input

Depending on how many cells you have, your sample may be low input or standard input. This classification depends on the type of bacteria and NEB provides guidelines for E.coli (standard: 1 x 10⁹ – 5 x 10⁹ cells; low: 5 x 10⁸ – < 1 x 10⁹ cells )and B.Cerus (low: 2 x 10⁸ – 4 x 10⁸ cells). See How to quantify bacterial cultures - From CFU and OD to counting chamber for tips on estimation.

Gram staining

If possible, you should determine the Gram classification of your samples. See Gram Stain Protocols from the American Society for Microbiology. If you are unsure you will have to work by trial and error. We recommend following the Gram negative protocol and observing carefully the efficency of your lysis.

Pellet bacterial cells in a Monarch Pestle Tube by centrifugation at maximum speed (> 12,000 x g) for 1 minute.
Follow the appropriate lysis protocol depending on the type of bacteria:

Gram-negative bacteriaGram-positive bacteria

a. Resuspend pellet in 300 µl (Low Input: 150 µl) cold PBS. Cold TE or Tris buffer may be used in place of PBS if preferred.

b. Add 10 µl Lysozyme (25 mg/ml, not provided) and mix by vortexing briefly.

c. Add 300 µl (Low Input: 150 µl) HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5-10 times.

d. Incubate at 37°C in a thermal mixer with agitation at the desired speed or incubate in a bath or heatbock ocassionally inverting to mix. The speed of the thermal mixer influences fragment length and lysis time. For most applications, maximum agitation speed (1400–2000 rpm) is recommended. For maximum gDNA size, agitate at 500 rpm. Incubation is complete when lysate turns clear, which is approximately 3–5 minutes for E. coli. At 500 rpm, lysis may take longer.

Tip

Thorough mixing is essential, especially with viscous samples.

a. Resuspend pellet in 300 µl (Low Input: 150 µl) of an appropriate lysis buffer containing a lytic enzyme and mix by vortexing briefly. STET buffer with freshly added lysozyme (10 mg/ml) works well for some Bacillus species.

b. Incubate at 37°C for 30 minutes (no agitation).

c. Add 300 µl (Low Input: 150 µl) HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5–10 times.
If working with a single thermal mixer, increase the temperature to 56°C. Following lysozyme treatment at 37°C, increase the temperature of the block in the thermal mixer to 56°C.
Add 20 µl (Low Input: 10 µl) of Proteinase K and mix by inverting 10–20 times.
Homogenization can be carried out using one of two methods, depending on the desired gDNA size: in a thermal mixer or with a rotor-stator homogenizer. If using a rotor-stator homogenizer, the sample must be in a 2 ml tube (not provided).

Thermal Mixer (for gDNA 50 kb up to ≥ 500 kb)Rotor-stator Homogenizer (for gDNA 50–250 kb)Needle shear

a. Incubate at 56°C for 30 minutes in a thermal mixer at the desired speed. The speed of the thermal mixer influences fragment length and lysis; higher agitation speeds reduce DNA size and sample lysis time. For most applications, including the standard ligation-based Oxford Nanopore Technologies (ONT) sequencing protocols, maximum agitation speed (1400–2000 rpm) is recommended to produce DNA fragments predominantly 50–250 kb.

To achieve maximum gDNA size, up to the Mb range, use a low agitation speed. Agitation at speeds < 500 rpm is not recommended as gDNA will be significantly tangled, which reduces the efficiency of protein removal in later steps. This tangled DNA is also difficult to dissolve during lysis and elution and can result in visible DNA aggregates.

a. Within a 2 ml tube, insert the tip of the homogenizer probe and turn on to the lowest setting. Homogenize 5–15 seconds; stop when foam begins to form in the lysate. Additional homogenization may be required to reach optimal gDNA size. gDNA size can be verified by pulsed field gel electrophoresis or FEMTO Pulse. Rotor-stator homogenizers may run at higher speeds after extended use; reduce homogenization time if necessary.

a. A low-cost substitute for the homogenizer, you can pass the sample through a 25G needle. Draw up and expel the bacterial lysate 5-10 times. You will have to characterize by trial and error your fragment size. Samples may be very viscous; you may shear with a pipette first.

Sharps Safety

Because the use of sharpes is involve, extra caution is advised. You can skip this step all together if you do not have other options.
Add 10 µl (Low Input: 5 µl) of RNase A and mix by inverting 5–10 times. Incubate for 10 minutes at 56°C with agitation in a thermal mixer at the speed used in Step 5. If you are not using a thermal mixer, incubate at 56°C with ocassional agitation by hand.
Change the heat block in the thermal mixer to accommodate a 2 ml tube, and preheat the block to 56°C. If a 2 ml tube block is not available, continue working with the 1.5 ml block.
Add 300 µl (Low Input: 150 µl) of Protein Separation Solution. Mix by inverting for 1 minute. Alternatively, a vertical rotating mixer at 20 rpm can be used.
Centrifuge for 10 minutes at 16,000 x g. If working with multiple samples, during centrifugation, prepare the plastics for Part 2, as indicated in the following step. The sample will separate into a large, clear upper phase (DNA) and a lower, clear phase (protein, usually on the bottom of the tube, but occasionally floating). There may also be a white precipitate at the bottom of the tube. Additional centrifugation time (10-20 minutes) may be required for complete phase separation, particularly when low agitation speeds were used.
If working with multiple samples, prepare and label the plastics for the upcoming steps. Each sample will require:
- Monarch Collection Tube II (no need to label)
- 1 Monarch Bead Retainer inserted into the collection tube; this will be used to remove the wash buffer from the gDNA bound to the beads.
- 2 Monarch 2 ml Tubes; one for phase separation and one for elution.
- 1 1.5 ml microfuge tube (DNA low bind recommended, not provided); this will be used to collect the eluate.
Using a 1000 µl (Low Input: 200 µl) wide-bore pipette tip, transfer the upper phase containing the DNA (large, clear phase) to a labeled Monarch 2 ml Tube. A substantial fraction of HMW DNA will be located at the interface between the clear upper phase and the protein phase; highest yields will be achieved by transferring as much of the upper phase as possible. Using a 200 µl wide-bore pipette tip to transfer the final volume of the upper phase is recommended for maximum yield. Avoid transferring material from the protein layer, though a small amount (1–2 µl) will not be detrimental. If protein enters the pipette tip, gently push it back into the tube. If a lower protein phase is not visible, leave ~30 µl behind to ensure protein is not carried over. Typically, the transferred volume will be ~ 800 µl (Low Input: ~400 µl). If the volume of the sample is < 700 µl (Low Input: < 350 µl), adjust the volume of isopropanol used in Step 2 of Part 2: HMW gDNA Binding and Elution to 0.7 volumes.

Pause point

If needed, you could keep samples at 4°C before precipitation with isopropanol in the next step.

DNA Isolation (Part II) — Binding and elution

Goal: Bind the high molecular weight DNA to the Monarch glass beads and elute and solubilize the DNA.

Video: Educator's Guide to Nanopore—Microbial Isolates: DNA Isolation II (Binding and Elution)

Using clean forceps, add 2 DNA Capture Beads to each sample, which should be contained in a Monarch 2 ml Tube.
Add 550 µl (Low Input: 275 µl) isopropanol, close the cap, and mix on a vertical rotating mixer at 10 rpm for 5 minutes to attach DNA to the beads. If a vertical rotating mixer is not available, invert slowly and gently by hand 25–30 times. A manual inversion is complete when the tube returns to the upright position. Slow inversion is critical for the DNA to bind to the beads; each full inversion should take ~5–6 seconds. If necessary, flick the tube to release any beads that stick to the bottom of the tube.
- After a 2–3 inversions, the solution becomes more viscous and the DNA will wrap loosely around the beads. During the following inversions, precipitation of gDNA may be visible, especially with larger input samples. The DNA complex will often contain small air bubbles. With increasing number of inversions, the DNA will completely wrap around the beads, often causing the beads to stick together. DNA binding to the beads should be complete after 25–30 inversions, and the solution should no longer be viscous. Additional inversions may be necessary for larger input samples.
Remove and discard liquid by pipetting. Avoid removing any of the gDNA wrapped around the glass beads. For optimal DNA solubility, avoid letting the bound DNA dry out on the beads during this and the following steps; add the next buffer quickly. There are two suggested options for carrying out this step:
- Keeping tube upright, insert pipette tip and gently push beads aside to remove liquid.
- Angle the tube so that beads remain at the bottom, and liquid reaches toward tube opening. Pipette from the liquid surface and continue to angle as liquid is removed (tube will be almost horizontal at the end).
  
  Figure on liquid removal—from NEB protocol
Add 500 µl gDNA Wash Buffer, close the cap, and mix by inverting the tube 2–3 times. Remove the gDNA Wash Buffer as described in step 3. The loose gDNA complex will condense around the beads more tightly.
Repeat the wash in Step 4 and remove the gDNA Wash Buffer by pipetting. Alternatively, the buffer can be removed by decanting: position a pipette tip at the top of the angled tube to prevent the beads from falling out. It is not necessary to remove all the gDNA Wash Buffer at this point.

Figure on liquid removal—from NEB protocol
Place a labeled bead retainer into a Monarch Collection Tube II. Pour the beads into the bead retainer and close the cap. Discard the used Monarch 2 ml Tube. When working with multiple samples, be sure to close the cap of the bead retainer after each transfer of beads.

Figure on bead removal—from NEB protocol
Optional

Optional bead binding workflow

In Protocol Guidance for Extraction of Ultra-High Molecular Weight (UHMW) Genomic DNA for Ultra-Long (UL) Read NGS Sequencing applications in Oxford Nanopore Technologies® workflows, the following steps are suggested:

The following alternative bead binding workflow is an option for experienced users looking for improved sequencing metrics. Invert the sample via slow manual inversion (~5 seconds per full inversion). Stop inverting once the DNA forms a loose “jelly” around the beads and the viscosity of the solution has returned to normal levels. This usually takes 20-25 inversions for cell and blood samples, and ~40 inversions for tissue samples. By keeping the DNA in this loose form, it is less compact and will therefore go into solution easier at the end of the purification process, leading to better results during the library prep and sequencing. However, when removing the supernatant after bead binding, care should be taken not to remove part of the DNA jelly, as it can easily slip into the pipette tip. If necessary, leave some of the liquid behind.

Upon adding the wash buffer, the DNA “jelly” will contract tighter around the beads and the sample will be easier to handle. Do not spin the beads to dry them after the wash when using this alternative workflow, as some DNA may detach from the beads. Instead, pour the beads containing the loose DNA complex into the bead retainer and tap the bead retainer gently on absorbent paper to remove traces of wash buffer. The DNA will not be completely dried, but that is not necessary at this stage; it will lead to better solubility upon elution. Do not expose the DNA to air longer than necessary; after tapping, pour the beads immediately into the elution buffer EB+, and return the empty bead retainer into the collection tube. Spin briefly in a benchtop minifuge to remove traces of wash buffer from the bead retainer walls. The dried bead retainer can now be placed in the DNA low bind tube that is required for the elution step.

Elution

Using the elution buffer-Triton mix (EB+) instead of Monarch Elution Buffer II results in better UL library prep and sequencing results. Instructions for formulating this mix are provided in “Important Notes Before Extraction.”

After the washes and dry spinning step, the following steps should be employed for elution:
1. Immediately pour the dried beads into the 2 ml tube with the pre-aliquoted elution buffer-Triton Mix (EB+).
2. Incubate 10 minutes at 56°C to elute the DNA off the beads. To facilitate elution during incubation, gently pipette and dispense the eluate a few times over the glass beads using a wide bore pipette tip; ensure that the DNA has been released from the beads (the viscosity of the eluate will increase).
3. If time is not limiting, let the eluate sit for at least 1 hour or overnight on the beads at room temperature before proceeding with the next step. This will improve elution efficiency and support effective resuspension of the UHMW DNA.
4. Pour the beads with the elution buffer into the bead retainer sitting in a DNA low bind 1.5 ml tube.
5. Centrifuge for 1 minute at maximum speed (>16,000 x g). Check if DNA was completely released from the beads. If after the spin DNA threads are visible between beads and eluate, centrifuge for 1 additional minute at max speed.
6. Using a wide bore pipette tip, carefully pipette eluate up and down 5 times for homogenization and incubate the samples for 5-10 minutes at 37°C. If needed, repeat the careful pipetting several times to resuspend the DNA. Store at 4°C or continue with dilution and resuspension guidance below.
UHMW DNA Dilution & Resuspension for Library Preparation

Samples should be further diluted to reach the optimal concentration for UL library preparation. Dilution should be done with the elution buffer-Triton mix that was used for elution, following the guidance below:
1. Add 560 µl elution buffer-Triton mix (EB+) to the sample to bring the total volume to 760 µl. Use 185 µl if working with MinION flow cells, for a total volume of 385 µl.
2. Pipette up and down 5-10 times using a P1000 wide-bore tip to resuspend the DNA.
3. Incubate the samples for 5-10 minutes at 37°C.
4. Repeat the steps 2 and 3 one or two times if samples do not appear homogeneous. Samples can be kept overnight at room temperature to support homogenization of the UHMW DNA or can be stored at 4°C. Once homogenized, take a 10 µl aliquot for quantitation2. Make sure DNA is well dissolved and no DNA clots are visible before proceeding with the Ultra Long library prep, Part 3 - Library Preparation. It is important to note that allowing the DNA at least 1 day to go into solution completely and “relax” typically leads to better library prep results.
Pulse spin (≤ 1 second) the sample in a benchtop minicentrifuge to remove any residual wash buffer from the beads.
Separate the bead retainer from the collection tube, pour the beads into a new, labeled Monarch 2 ml Tube, and insert the used bead retainer into the labeled 1.5 ml microfuge tube (DNA low bind recommended, not provided) for later use during elution. Discard the used collection tube.
Immediately add 100 µl Elution Buffer II onto the glass beads and incubate for a minimum of 5 minutes at 56°C in a thermal mixer with agitation at the lowest speed (300 rpm). Halfway through the incubation, ensure the beads are not stuck to the bottom of the tube by tilting the tube almost horizontally and gently shaking. This ensures that the beads can move freely, allowing for optimal release of the DNA from the beads. It also ensures that the lower bead does not stick to the bottom of the tube during the following transfer step. Elution volume can be reduced to as low as 50 μl without affecting recovery. However, if using < 100 μl, the gentle shaking of the sample should be done several times during the incubation to ensure complete wetting of the beads.

Figure capturing DNA on beads—from NEB protocol
Ensure the bead retainer is inserted into the 1.5 ml microfuge tube. Pour the eluate and the glass beads into the bead retainer and close the cap. When working with more than 1 sample, it is important to close the cap after each transfer of beads. Typically, all the eluate flows into the bead retainer upon pouring. If any volume remains in the 2 ml tube, spin briefly and transfer.

Figure eluting DNA from beads—from NEB protocol
Centrifuge for 30 seconds at 12,000 x g to separate the eluate from the glass beads. Discard the beads and retainer.
Pipette eluate up and down 5–10 times with a wide bore pipette tip and ensure any visible DNA aggregates are dispersed. Before analysis or downstream use, HMW DNA must be homogeneously dissolved. After pipetting, incubate at 37°C for 30-60 minutes, overnight at room temperature, or for > 24 hours at 4°C. Pipette up and down 5-10 times again before analyzing or using the HMW DNA. Samples processed using low agitation speeds during lysis will require additional time to fully dissolve. See additional guidance in “Homogenization of HMW DNA Samples”. Samples can be stored at 4°C for short term use (weeks), or at -20°C for long term storage. The elution buffer (10 mM Tris, pH 9.0, 0.5 mM EDTA) is formulated for long term storage of gDNA.

DNA quantification with Qubit™

Goal: Determine the yeild of your DNA extraction.

Video: Educator's Guide to Nanopore—Microbial Isolates: DNA quantification with Qubit)

References

This protocol is from the Themofisher manual.

Set up and label the required number of Qubit™ tubes for standards and samples. The Qubit™ dsDNA HS Assay requires 2 standards.
Use the provided assay tubes.

Use the working solution to prepare the standards and samples as follows:

	Standard Assay Tubes	User Sample Assay Tubes
Volume of working solution	190 μL	180–199 μL
Volume of standard	10 μL	—
Volume of user sample	—	1–20 μL
Total volume in each assay tube	200 μL	200 μL

We recommend using 1-2 μL of the prepared library.

Vigorously vortex for 3–5 seconds. Be careful not to create bubbles
Allow all tubes to incubate at room temperature for 2 minutes, then proceed to read standards and samples
On the Home screen, touch dsDNA, then select dsDNA High Sensitivity as the assay type. Touch Read standards to proceed.

Note

If you have already performed a calibration for the selected assay, the instrument prompts you to choose between reading new standards and running samples using the previous calibration. If you want to use the previous calibration, skip to step 8. Otherwise, continue with step 6.
Insert the tube containing Standard #1 into the sample chamber, close the lid, then touch Read standard. When the reading is complete (~3 seconds), remove Standard #1.
Insert the tube containing Standard #2 into the sample chamber, close the lid, then touch Read standard. When the reading is complete, remove Standard #2.

Note

The instrument displays the results on the Read Standards screen. For information on interpreting the calibration results, refer to the Qubit™ 4 Fluorometer User Guide.
Touch Run samples.
On the assay screen, select the Sample volume and units.

a. Touch the + or – buttons on the wheel, or anywhere on the wheel itself, to select the sample volume added to the assay tube (1–20 μL).

b. From the Unit dropdown menu, select the units for the output sample concentration.
Insert a sample tube into the sample chamber, close the lid, then touch Read tube. When the reading is complete (~3 seconds), remove the sample tube. The top value (in large font) is the concentration of the original sample and the bottom value is the dilution concentration. For information on interpreting the sample results, refer to the Qubit™ 4 Fluorometer User Guide.
Repeat step 11 until all samples have been read.

Nanopore

Complete a flow cell check to assess the number of pores available. This should be done on the day you plan to load the library.

Library Preperation — DNA end repair and barcoding

Goal: Prepare DNA ends and attaching barcode sequences.

Video: Educator's Guide to Nanopore—Microbial Isolates: Library prep – DNA end repair and barcoding

Nanopore

This protocol follows the MinION version of Ligation sequencing gDNA - Native Barcoding Kit 24 V14 (SQK-NBD114.24) from Oxford Nanopore.

Oxford Nanopore's protocol provides the following overview of the library preparation workflow.

Figure capturing native ligation workflow—from ONT protocol

DNA repair and end-prep

Note

For samples containing long gDNA fragments, we recommend using wide-bore pipette tips for the mixing steps to preserve the DNA length.

Thaw the AMPure XP Beads (AXP) and DNA Control Sample (DCS) at room temperature and mix by vortexing. Keep the beads at room temperature and store the DNA Control Sample (DCS) on ice.
Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
Tip

For optimal performance, NEB recommends the following:

a. Thaw all reagents on ice.

b. Flick and/or invert the reagent tubes to ensure they are well mixed.

Note

Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.

c. Always spin down tubes before opening for the first time each day.

d. The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note
- It is important the buffers are mixed well by vortexing.
- The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
Warning
- Do not vortex the NEBNext FFPE DNA Repair Mix or NEBNext Ultra II End Prep Enzyme Mix.
- It is important that the NEBNext FFPE DNA Repair Buffer and NEBNext Ultra II End Prep Reaction Buffer are mixed well by vortexing.
- Check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise any precipitate.
Dilute your DNA Control Sample (DCS) by adding 105 µl Elution Buffer (EB) directly to one DCS tube. Mix gently by pipetting and spin down.
Tip
- One tube of diluted DNA Control Sample (DCS) is enough for 140 samples. Excess can be stored at -20°C in the freezer.
- We recommend using the DNA Control Sample (DCS) in your library prep for troubleshooting purposes. However, you can omit this step and make up the extra 1 µl with your sample DNA.
In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), prepare your DNA samples:
For >4 barcodes, aliquot 400 ng per sample
For ≤4 barcodes, aliquot 1000 ng per sample
Make up each sample to 11 µl using nuclease-free water. Mix gently by pipetting and spin down.

Combine the following components per tube/well; Between each addition, pipette mix 10 - 20 times;

✓	Reagent	Volume
	DNA sample	11 µL
	Diluted DNA Control Sample (DCS)	1 µL
	NEBNext FFPE DNA Repair Buffer	0.875 µL
	Ultra II End-prep Reaction Buffer	0.875 μL
	Ultra II End-prep Enzyme Mix	0.75 μL
	NEBNext FFPE DNA Repair Mix	0.5 µL

Info

We recommend making up a mastermix of the end-prep and DNA repair reagents for the total number of samples and adding 3 µl to each well.

Classroom

You can prepare the mastermix and aliquot to students.

Ensure the components are thoroughly mixed by pipetting and spin down in a centrifuge.
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
Resuspend the AMPure XP beads (AXP) by vortexing.
Add 15 µl of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature or agitate by hand intermittently over the duration of the incubation.
Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
Spin down the samples and pellet the beads on a magnet until the eluate is clear and colorless. Keep the tubes on the magnet and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Tip

If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repeat the previous step.
Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colorless.
Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Tip

Dispose of the pelleted beads.

Optional

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

Pause point

Take forward an equimolar mass of each sample to be barcoded forward into the native barcode ligation step. However, you may store the samples at 4°C overnight.

Library Preperation — Pooling, sequencing adapters, and cleaning

Goal: Barcode end-preped DNA and add sequencing adapters.

Video: Educator's Guide to Nanopore—Microbial Isolates: Library prep – Pool and clean

Native barcode ligation

Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:

a. Thaw the reagents at room temperature.

b. Spin down the reagent tubes for 5 seconds.

c. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Thaw the EDTA at room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the Native Barcodes (NB01-24) at room temperature. Briefly spin down, individually mix the barcodes required for your number of samples by pipetting, and place them on ice.
Select a unique barcode for each sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment.

Note

Only use one barcode per sample.
In clean 0.2 ml PCR-tubes or a 96-well plate, add the reagents in the following order per well; Between each addition, pipette mix 10 - 20 times.
✓ Reagent Volume
End-prepped DNA

7.5 µl
Native Barcode (NB01-24)

2.5 µl
Blunt/TA Ligase Master Mix

10 µl
Total 20 µl
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate for 20 minutes at room temperature.
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
Note

Ensure you follow the instructions for the cap color of your EDTA tube. EDTA is added at this step to stop the reaction.
✓ EDTA cap color Volume
For clear cap EDTA 2 µl
For blue cap EDTA 4 µl

Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.

Note

Ensure you follow the instructions for the cap colour of your EDTA tube.

✓	Volume per sample	For 6 samples	For 12 samples	For 24 samples
	Total volume for preps using clear cap EDTA	22 µl	132 µl	264 µl
	Total volume for preps using blue cap EDTA	24 µl	144 µl	288 µl

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add 0.4X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.

Note

Ensure you follow the instructions for the cap color of your EDTA tube.

✓	Volume per sample	For 6 samples	For 12 samples	For 24 samples
	Volume of AXP for preps using clear cap EDTA	9 µl	53 µl	106 µl
	Volume of AXP for preps using blue cap EDTA	10 µl	58 µl	115 µl

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colorless, and pipette off the supernatant.
Keep the tube on the magnetic rack and wash the beads with 700 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Note

If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repeat the previous step.
Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 35 µl nuclease-free water by gently flicking.
Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnetic rack until the eluate is clear and colorless.
Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Optional

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

Pause point

Take forward an equimolar mass of each sample to be barcoded forward into the native barcode ligation step. However, you may store the samples at 4°C overnight.

Adapter ligation and clean-up

Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:

a. Thaw the reagents at room temperature.

b. Spin down the reagent tubes for 5 seconds.

c. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Note
- Do NOT vortex the Quick T4 DNA Ligase.
- The NEBNext Quick Ligation Reaction Buffer (5x) may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.
Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
Note

Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally.
- To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB)
- To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)
We recommend using the Long Fragment Buffer (LFB)
Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and keep at room temperature.
In a 1.5 ml Eppendorf LoBind tube, mix in the following order; Between each addition, pipette mix 10 - 20 times.
✓ Reagent Volume
Pooled barcoded sample

30 µl
Native Adapter (NA)

5 µl
NEBNext Quick Ligation Reaction Buffer (5X)

10 µl
Quick T4 DNA Ligase

5 µl
Total 50 µl
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 20 minutes at room temperature.

Note

The next clean-up step uses Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) rather than 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 20 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
Wash the beads by adding either 125 μl Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
Remove the tube from the magnetic rack and resuspend pellet in 15 µl Elution Buffer (EB).
Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.
Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube; dispose of the pelleted beads.

Optional

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

Depending on your DNA library fragment size, prepare your final library in 12 µl of Elution Buffer (EB).

Fragment library length Flow cell loading amount

Very short (<1 kb) 100 fmol

Short (1-10 kb) 35–50 fmol

Long (>10 kb) 300 ng

Note

If the library yields are below the input recommendations, load the entire library.
If required, we recommend using a mass to mol calculator such as the NEB calculator.
We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short-term storage or repeated use, for example, re-loading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes.
If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Depending on how many flow cells the library will be split across, more Elution Buffer (EB) than what is supplied in the kit will be required.

Pause point

Take forward an equimolar mass of each sample to be barcoded forward into the native barcode ligation step. However, you may store the samples at 4°C overnight.

Preparing and loading flow cell

Goal:Prime the flow cell and load the sequencing library.

Video: Educator's Guide to Nanopore—Microbial Isolates: Preparing and Loading Flow Cell

Note

Using the Library Solution

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.
Note

Oxford Nanopore is in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
- Single-use tubes format: Add 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml and 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).
- Bottle format: In a suitable tube for the number of flow cells, combine the following reagents:
✓ Reagent Volume per flow cell
Flow Cell Flush (FCF) 1,170 µl
Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl
Flow Cell Tether (FCT) 30 µl
Total volume 1,205 µl
Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the priming port cover to ensure correct thermal and electrical contact.
Slide the flow cell priming port cover clockwise to open the priming port.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:

a. Set a P1000 pipette to 200 µl

b. Insert the tip into the priming port

c. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip

Warning

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.

Visually check that there is continuous buffer from the priming port across the sensor array.

Figure demonstrating removal of buffer from flow cell—from ONT protocol
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.

Tip

The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

Oxford Nanopore recommends using the Library Beads (LIB) for most sequencing experiments. However, the Library Solution (LIS) is available for more viscous libraries.

In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:

✓	Reagent	Volume per flow cell
	Sequencing Buffer (SB)	37.5 µl
	Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using	25.5 µl
	DNA library	12 µl
	Total volume	75 µl

Complete the flow cell priming:

a. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

b. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
Place the light shield onto the flow cell, as follows:

a. Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.

b. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.

Note

Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.

Oxford Nanopore recommends leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.
Close the device lid and set up a sequencing run on MinKNOW.

Flow cell check, mid-run check-in, and final output review

Goal: Run the flow cell using MinKNOW software.

Video: Educator's Guide to Nanopore—Microbial Isolates: Flow Cell Check, Run, and Output Review

Flow cell check

Navigate to the Start page and select 'Flow cell check' to open the flow cell check page.
When the MinION Flow Cell type and flow cell ID have been recognized, click 'Start' to begin.

Nanopore

See additional details: Flow cell check.

Sequencing run

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.
Type in the experiment name, sample ID and choose flow cell type from the drop down menu.
Select the kit and expansion(s) used to prepare the library. (Native Barcoding Kit 24 V14 (SQK-NBD114.24))
Choose your basecalling options; We recommend:
- Fast basecalling for most situations.
- Modified bases is off
Choose your barcoding options; should be ON.
Choose your alignment options; should be OFF.
Select the output options: We recommend
- FASTQ: Checked on
- POD5: Checked on
Click 'Start' to run the experiment.

Nanopore

See additional details: Starting a sequencing run on MinION Mk1B/Mk1D.

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Ended: Annotated Experiments

Bioinformatics ↵

Data Organization and Management in Nanopore Sequencing

The importance of good data management

Effective data management is essential for ensuring the integrity, reproducibility, and long-term accessibility of sequencing data. Following best practices for organizing, storing, and documenting sequencing data benefits both research efficiency and educational experiences. In genomics, the volume of data generated can be substantial, making structured data organization critical.

A guiding framework for good data stewardship is the FAIR Principles:

Findable – Data should be easily discoverable using standardized metadata.
Accessible – Data should be retrievable via established repositories and protocols.
Interoperable – Data should be compatible with various analysis tools and software.
Reusable – Data should be well-documented and structured to allow for future use.

For educators, introducing students to data hygiene—including proper file naming, directory structuring, and metadata collection—helps instill best practices that are critical for careers in bioinformatics, computational biology, and genomics.

Instruction tip

Managing Student-Generated Data

Managing student-generated data in a Nanopore sequencing course requires careful planning to ensure smooth data sharing, organization, and long-term usability. Educators should establish a clear data-sharing strategy that facilitates seamless file exchange between instructors and students, particularly given the large file sizes and/or large number of files associated with sequencing data. Cloud storage solutions like CyVerse, Google Drive, or institutional servers may be useful for storing and distributing datasets, but file size limitations and access controls should be considered. Additionally, educators should anticipate the scientific value of student-generated data—insisting on good data practices from the beginning can ensure that sequencing results are valid, reproducible, and valuable in aggregate.

Developing a classroom data management plan can help structure how data are named, stored, and tracked across multiple course sections, lab groups, or project days. Establishing standardized file-naming conventions that incorporate identifiers such as course section, date, group number, and experiment type will allow educators to efficiently organize and retrieve data. Collecting consistent metadata across student projects is also essential if data are to be meaningfully analyzed or aggregated for further research or public sharing. Resources such as the FAIR Principles provide guidance on making data findable, accessible, interoperable, and reusable.

File and object names and metadata to keep track of from the start

Project name: At the most encompassing level of organization, have a simple project name
Students involved: Have a way of tracking students by more than give names and initials. Perhaps all students can be assigned a short unique identifier. You could consider having students create and use ORCIDs.
Sample names: Have a way to name specific samples. The sample name might also include or be linked to acquisition information such as the date collected, or the location of sampling. If a single sampled is sampled multiple times, have a way to track and indicate this.
Replicates: Have a way to indicate replication.
Data directory structures: Consider how directories on storage platforms will be organized. Have rules for how folders should be named, when folders should be created, and what should be in them.
Collision avoidance: Some files and folders will not have unique names. For example, every time you run a 16S workflow on MinKNOW, you will get folders named barcode01, barcode02,barcode03... Know when you will encounter these and have a way to manage their organization, or if needed, renaming, to avoid confusion.
Software management: Have a way to keep track of the software versions of any software you will use.

While this list is not exhaustive, considering these possibilities ahead of time will make for smooth organization later.

By integrating good data hygiene and documentation practices, educators can help students engage with sequencing workflows in a way that mirrors professional research environments.

Data management plans

A Data Management Plan (DMP) is an essential document that outlines how data will be:

Collected
Stored and backed up
Processed and analyzed
Shared or archived

Many funding agencies require DMPs to ensure that research data remains well-documented and reusable. While formal DMPs may not be necessary for classroom projects, educators should emphasize the importance of:

Keeping consistent directory structures
Using clear, standardized file naming conventions
Documenting key metadata
Implementing proper backup procedures

Additional Reading

National Human Genome Research Institute. (n.d.). Genomic Data Science. National Human Genome Research Institute (NHGRI).
Oza, V. H., Whitlock, J. H., Wilk, E. J., Uno-Antonison, A., Wilk, B., Gajapathy, M., Howton, T. C., Trull, A., Ianov, L., Worthey, E. A., & Lasseigne, B. N. (2023). Ten simple rules for using public biological data for your research. PLoS Computational Biology, 19(1), e1010749. https://doi.org/10.1371/journal.pcbi.1010749.
Form, D., & Lewitter, F. (2011). Ten Simple Rules for Teaching Bioinformatics at the High School Level. PLoS Computational Biology, 7(10), e1002243. https://doi.org/10.1371/journal.pcbi.1002243.
Noble, W. S. (2009). A Quick Guide to Organizing Computational Biology Projects. PLoS Computational Biology, 5(7), e1000424. https://doi.org/10.1371/journal.pcbi.1000424.

Common file types in Nanopore sequencing

Nanopore sequencing generates multiple types of data files at different stages of the workflow. Below is an overview of the key file formats:

Raw data

POD5 (.pod5) [Documentation]
Current standard format for storing raw signal data from Nanopore sequencing. Replaces the older FAST5 format for improved performance and scalability.
FAST5 (.fast5) [Legacy]
Used in older Nanopore sequencing workflows to store raw signal data. This format is no longer in use, having been replaced by POD5.

Processed data

FASTQ (.fastq) [Documentation]
Primary file format for storing basecalled sequence reads, including nucleotide sequences and associated quality scores.
BAM (.bam) / CRAM (.cram) [Documentation]
Binary formats used to store sequence alignments to a reference genome. CRAM is a more compressed version of BAM.

Reference data

FASTA (.fasta / .fa) [Documentation]
Stores nucleotide or protein sequences without quality scores. Often used for reference genomes or consensus sequences.
GFF/GTF (.gff / .gtf) [Documentation]
Contains genome annotations, such as gene locations and functional elements.

Analysis and metadata

CSV/TSV (.csv / .tsv)
Tabular files commonly used for summarizing sequencing statistics, barcode classifications, or other metadata.
JSON (.json)
Structured format for storing run configurations, analysis parameters, or experiment details.
VCF (.vcf)
Variant Call Format, used for storing genetic variations detected in sequencing data.

Reports

HTML (.html)
Many analysis pipelines generate interactive reports in HTML format, offering visual summaries, charts, and metrics. This format is useful for sharing final results with collaborators or for archiving purposes.

Managing genomics data for long-term use

Metadata collection

Metadata provides essential context for sequencing datasets and is often required for submitting data to public repositories like the NCBI Sequence Read Archive (SRA). Key metadata elements may include:

Sample ID (Unique identifier for the sample)
Collection date (When the sample was obtained)
Location (Geographic origin of the sample)
Extraction and library prep methods (Protocols used)
Sequencing platform and flow cell type
Bioinformatics processing steps (Pipeline details, tools used)

Storage and backup strategies

Primary Storage: A local high-capacity SSD (recommended 1TB or more) for sequencing and short-term analysis.
Backup Storage: Redundant copies using external SSDs or network drives.
Cloud Solutions: Platforms like Google Drive, CyVerse, or JetStream2 for scalable storage.
Public Repositories: Depositing finalized datasets in SRA, EMBL-EBI ENA, or MG-RAST for community access.

Preparing data for public submission

Ensure FASTQ files are properly formatted.
Validate metadata against repository requirements.
Use consistent file naming conventions (e.g., sampleID_experimentType_date.fastq).
Include a README file describing dataset contents and processing steps.

Quickstart: How to find and download Nanopore reads from the Sequence Read Archive (SRA)

The NCBI Sequence Read Archive (SRA) is a public repository that stores sequencing data, including Oxford Nanopore sequencing reads. This tutorial provides a step-by-step guide to searching for data, in this case, Actinomyces sequences generated using Oxford Nanopore technology and downloading a FASTQ file for further analysis. You can adapt the tutorial to search for data from your organism of interest and use those data for reanalysis and to practice your bioinformatics skills.

Open your web browser and visit the NCBI SRA website.
Click on the Advanced link beneath the search bar to access the detailed search options.
In the first box of the search Builder, type Actinomyces and click the Show index list link. The number in parentheses shows how many records match the term. You can click on the term of choice, in this case actinomyces (936), and select Organism from the dropdown to ensure that is the level being searched.
In the second box of the search Builder, type Oxford Nanopore and click the Show index list link. The number in parentheses shows how many records match the term. You can click on the term of choice, in this case oxford nanopore (875940), and select Platform from the dropdown to ensure that is the level being searched.
Click the Search button to retrieve relevant sequencing datasets.
Browse through the search results and look for a 16S sequencing dataset. In this case, we choose a selection marked 16S rRNA sequence of isolates. Note the number of bases (M - megabases, G - gigabases) and size of the dataset in Mb/Gb is shown, alerting you to the size of the potential download.; Click on a result to view the dataset details.
The results page will have various metadata including an abstract for the entry which may contain useful background on the experiment that produced the data.

Tip

The All experiments or All runs link will help you find connected datasets which may have come from other libraries or samples in this same experiment.
Locate the Runs table, which lists individual sequencing runs associated with the dataset.
Click on the Run Accession link (e.g., SRRXXXXXXX) of a single sequencing run to open its details page.
On the run browser page, click the FASTA/FASTQ download link.
Under Download click on FASTQ to start a download for your file. The file will be in a compressed .fastq.gz format. This is a typical input for many downstream analyses.

Additional Notes

The downloaded FASTQ file contains raw sequence reads and quality scores that can be used for taxonomic classification, assembly, or other bioinformatics workflows.
Advanced users may explore SRA Toolkit, a command-line software package that enables more efficient searching, downloading, and processing of SRA data. The toolkit provides options such as prefetch for downloading and fastq-dump for extracting sequence reads. More details can be found in the SRA Toolkit documentation.

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Software Installation and Management

Installing essential software for Nanopore sequencing

To operate a Nanopore sequencer and analyze sequencing data, three key software tools are required:

MinKNOW: The primary control software for Oxford Nanopore sequencing devices.
EPI2ME: A cloud-based or local analysis platform for processing sequencing data.
Docker: A containerization platform required for running some local bioinformatics workflows.

Below are step-by-step instructions for downloading and installing each tool on macOS and Windows.

MinKNOW

MinKNOW is used to control Nanopore sequencing devices, perform basecalling, and monitor sequencing progress.

macOS InstallationWindows Installation

Visit the Oxford Nanopore Downloads Page and choose your device.
Select MacOS for Apple or Intel (open About This Mac, choose Apple menu  > About This Mac to check the Chip listed.); click Download.
Sign in to your Oxford Nanopore account (or create one if you don’t have an account); your download should begin.
Open the downloaded .dmg file and drag the MinKNOW application to the Applications folder.
Open MinKNOW from the Applications folder.
Follow the on-screen setup instructions and allow necessary permissions when prompted.
Restart your computer to ensure all components load correctly.

Visit the Oxford Nanopore Downloads Page and choose your device.
Sign in to your Oxford Nanopore account (or create one if you don’t have an account); your download should begin.
Download the latest MinKNOW for Windows.
Open the downloaded .exe file and follow the on-screen installation instructions.
Restart your computer if prompted.
Open MinKNOW and ensure it launches properly.

Installing EPI2ME

EPI2ME is a workflow management tool used for analyzing Nanopore sequencing data.

macOS InstallationWindows Installation

Visit the EPI2ME Labs download page.
Select macOS for M1/M2 (Apple) or Intel (open About This Mac, choose Apple menu  > About This Mac to check the Chip listed.); click Download.
Open the .dmg file and drag the EPI2ME application to the Applications folder.
Open EPI2ME from the Applications folder.
Sign in with an existing account or create a new account if needed.
Follow the initial setup process to ensure EPI2ME is ready for use.

Visit the EPI2ME Labs download page.
Download EPI2ME for Windows.
Open the downloaded .exe file and follow the on-screen installation instructions.
Launch EPI2ME and sign in with an existing account or create a new one.
Complete the initial setup and confirm that the software runs correctly.

Tip

EPI2ME requires the Window's Subsystem for Linux (WSL) please see these instructions for installation.

Installing Docker

Docker is required to run many EPI2ME workflows locally.

macOS InstallationWindows Installation

Visit the Docker website and scroll to find the Download Docker for Desktop button. Select Download for Mac MacOS for Apple Silicon or Intel Chip (open About This Mac, choose Apple menu  > About This Mac to check the Chip listed.).
Open the .dmg file and drag the Docker application to the Applications folder.
Open Docker from the Applications folder.
Follow the setup process and allow necessary permissions when prompted.
Once Docker is running, verify the installation by opening a terminal and running:

docker --version

Download Docker Desktop for Windows.
Open the downloaded .exe file and follow the setup instructions.
During installation, enable WSL 2 (Windows Subsystem for Linux) when prompted. If WSL 2 is not installed, follow the instructions provided by Docker to install it.
Restart your computer when the installation is complete.
Open Docker Desktop from the Start menu.
Verify the installation by opening Command Prompt and running:

docker --version

Other software packages for Nanopore sequencing and genomics analysis

Below is a curated list of 40 software tools that are widely used for Nanopore sequencing and genomics data analysis. The selection is based on recent updates in their documentation (within the past two years) and current usage in the field. Each entry includes a brief description and a link to the tool's documentation or repository. Some of these are more advanced in their usage (i.e., commandline) but you are likely to see them or want to learn more about their capabilities.

Below is a curated list of 40 software tools widely used for Nanopore sequencing and genomics data analysis. The tools are categorized based on their primary function to facilitate easy navigation.

Quality Control and Preprocessing

FastQC
Performs quality control checks on raw sequencing data in FASTQ format.
MultiQC
Aggregates reports from multiple QC tools into a single summary.
NanoPlot
Generates visualizations and statistics for assessing Nanopore sequencing data quality.
Seqtk
A lightweight tool for processing FASTA/FASTQ files, including format conversion and subsampling.

Basecalling and Signal Processing

Dorado
High-performance basecaller for Nanopore sequencing data.
Bonito
A deep learning-based basecaller for improved accuracy.
Nanopolish
Analyzes raw signal data to refine consensus sequences and detect base modifications.

Genome Assembly

Flye
A de novo assembler optimized for Nanopore and PacBio long reads. Probably the best place to start for assembly with Nanopore reads.
Canu
Assembles genomes using long-read sequencing data.
SPAdes
A versatile genome assembler that supports hybrid assemblies combining short and long reads.
Unicycler
A hybrid assembly pipeline combining short and long reads for high-quality microbial genome assemblies.

Genome Alignment

Minimap2
An efficient aligner for mapping long sequencing reads to a reference genome.
BWA-MEM2
A faster, more efficient version of BWA-MEM for read alignment.
STAR
A fast aligner optimized for RNA sequencing.

Variant Calling and Genome Polishing

Medaka
Polishes consensus sequences generated from Nanopore sequencing data.
Longshot
A variant caller optimized for long-read sequencing.
FreeBayes
A haplotype-based variant caller for detecting genetic variations.
DeepVariant
Uses deep learning to improve variant calling accuracy.
Pilon
Improves genome assemblies by correcting errors with short-read data.
Racon
A consensus module for polishing genome assemblies from long-read sequencing.

Taxonomic and Metagenomic Analysis

Kraken2
A taxonomic classifier that assigns microbial reads to taxa using k-mer-based algorithms.
MEGAN
Enables taxonomic and functional analysis of metagenomic data.
MetaPhlAn
A tool for profiling microbial communities from metagenomic data.

Genome Visualization and Annotation

IGV (Integrative Genomics Viewer)
A high-performance visualization tool for large-scale genomic data.
UCSC Genome Browser
A web-based genome browser for accessing and visualizing genomic annotations.
BEDTools
A toolkit for genome arithmetic and feature comparisons.
SnpEff
Annotates genetic variants and predicts their functional impact.
VCFtools
A collection of utilities for processing and analyzing Variant Call Format (VCF) files.
PLINK
A toolset for population-based genetic association studies.
QUAST
Evaluates and compares genome assemblies.

Workflow Management and Reproducibility

Nextflow
A workflow management system for scalable and reproducible bioinformatics pipelines.
Snakemake
A workflow engine for creating reproducible bioinformatics pipelines.
Bioconda
A Conda channel that simplifies installation of bioinformatics tools.
Galaxy
A web-based platform for running bioinformatics workflows.
CyVerse
A cloud-based data management and computation platform for large-scale genomics projects.

General-Purpose Computational Tools

Docker
A containerization platform for reproducible bioinformatics environments.
Jupyter Notebook
An interactive computing environment for running and sharing bioinformatics code.
RStudio
A popular IDE for R, widely used in bioinformatics and data visualization.
NCBI SRA Toolkit
A command-line toolset for accessing sequencing data from the NCBI Sequence Read Archive.
JetStream2
A cloud-based computing resource providing access to GPUs and bioinformatics tools.

For further reading see Awesome Bioinformatics.

Building more advanced bioinformatics skills – The Carpentries

The Carpentries is a global community dedicated to teaching foundational coding and data science skills through hands-on, interactive workshops. Their lessons are particularly valuable for researchers and educators looking to build computational skills for bioinformatics and genomics. The Software Carpentry and Data Carpentry initiatives offer structured lessons on command-line tools, version control, and programming languages like Python and R. Lessons are self-paced, and in-person workshops are available on every continent!

Additional Reading

The Genomics Data Carpentry lessons provide an excellent introduction to managing and analyzing sequencing data, covering topics such as the UNIX command line, working with FASTQ files, quality control, and genome assembly. These lessons are designed for beginners and offer an accessible entry point for educators and students looking to incorporate bioinformatics into their work.

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Cloud Computing and other Platforms ↵

DNA Subway: A User-Friendly Bioinformatics Platform

DNA Subway is an intuitive, web-based bioinformatics tool designed to make genomic data analysis accessible to students and educators. Developed by CyVerse, DNA Subway simplifies complex bioinformatics workflows, guiding users through DNA barcoding, genome annotation, and comparative genomics analyses. The platform allows users to analyze sequencing data without requiring advanced computational skills, making it ideal for classroom and research use.

Transition to DNA Subway 2.0

The current version of DNA Subway (DNA Subway 1.0) is sunsetting in June 2025, and will be replaced with DNA Subway 2.0, which is undergoing a complete redesign to better support Nanopore sequencing data analysis This next-generation platform will include:

Expanded DNA barcoding: Designed to handle data from Nanopore sequencing enabling educators and students to analyze their own barcode sequences.
Small genome analysis: Support for assembling and annotating microbial and other small genomes using Nanopore sequencing data.
Enhanced user experience: A more modern and interactive interface with improved workflow tracking and data visualization.
Cloud-Based computing: Streamlined data processing through JetStream2 computational **, reducing the need for local computing resources.

Learn More

For updates on the transition to DNA Subway 2.0 and new features, visit the DNA Subway Sunset Page. Educators and researchers interested in integrating DNA Subway into their curriculum can also explore DNA Subway Learning Resources for guides and learning materials.

Check back soon for tutorials!

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Jetstream2: Advanced Cloud Computing for Nanopore Sequencing

Jetstream2 is a cloud-based computing platform designed to provide researchers and educators with flexible, on-demand access to high-performance computing resources. It offers a user-friendly environment that simplifies data analysis, accelerates discovery, and enhances the availability of artificial intelligence (AI) tools across various scientific domains.

Bioinformatics

With training, anything is possible. That said, JetStream2 cloud resources require a fair bit of bioinformatics skill. Make use of their support and the Nanopore Network Slack community for help.

Introduction to Jetstream 2

Key Features of Jetstream2

Scalability: Jetstream2 delivers 8 petaFLOPS of virtual supercomputing power, allowing users to scale their computational resources according to project needs.
User-Friendly Interface: The platform provides an intuitive interface, enabling users to create virtual machines (VMs) that replicate your local computing environments.
Diverse Resource Allocation: Jetstream2 comprises four distinct resources:
Jetstream2 (CPU only): Standard computational tasks.
Jetstream2 Large Memory: Memory-intensive applications.
Jetstream2 GPU: Tasks requiring graphical processing units.
Jetstream2 Storage: Dedicated storage solutions.

Each resource is allocated individually, allowing users to tailor their environment to specific project requirements.

Advantages for Nanopore Sequencing

Nanopore sequencing generates substantial volumes of data and often requires intensive computational analysis. Jetstream2's infrastructure is particularly well-suited to meet these demands:

High-Performance Cloud Computing: The platform's substantial computational power accelerates data processing tasks such as basecalling, genome assembly, and variant analysis.
GPU Access: Jetstream2 offers access to GPU resources, which are essential for running advanced machine learning algorithms and accelerating specific bioinformatics applications. To utilize GPU instances, users must have explicit access to the Jetstream2-GPU resource.
Scalable Storage Solutions: With a default allocation of 1 TB of storage—expandable upon request—Jetstream2 efficiently manages the large datasets typical of Nanopore sequencing projects.

Getting Started with Jetstream2

To begin using Jetstream2, you will need an allocation (free) from NSF ACCESS-CI:

Utilize the Getting Started Guide for step-by-step instructions on accessing resources and creating your first VM instance.

For educators, ACCESS-CI offers educational allocations to support teaching initiatives. Detailed information on obtaining an allocation is available on the ACCESS-CI website. Once granted, these allocation credits can be applied to Jetstream2, providing access to its comprehensive computational resources.

By leveraging Jetstream2, researchers and educators can efficiently manage and analyze large-scale Nanopore sequencing data, facilitating advanced bioinformatics research and education.

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Amazon Web Services: A Comprehensive Cloud Platform for Genomics

Amazon Web Services (AWS) is a leading cloud computing platform offering a vast array of services that provide flexible, scalable, and reliable computing solutions. For advanced users in genomics, AWS presents powerful tools to manage, process, and analyze large-scale sequencing data. However, AWS is best suited for highly experienced users who are comfortable with cloud computing concepts, resource management, and cost monitoring.

Key AWS Services for Genomics

Amazon Elastic Compute Cloud (EC2): EC2 provides resizable virtual machines, allowing users to run applications on a secure and scalable infrastructure. In genomics, EC2 instances can be configured with high-performance computing capabilities, including access to Graphics Processing Units (GPUs), which are essential for computationally intensive tasks such as genome assembly, basecalling, and variant analysis.
Amazon Simple Storage Service (S3): S3 offers scalable object storage designed for large volumes of data. Genomics researchers can utilize S3 buckets to store raw sequencing data, intermediate files, and final analysis results. The service ensures high durability and availability, making it suitable for the extensive datasets generated by sequencing projects.

Considerations and Getting Started

While AWS provides robust infrastructure, it is essential to be mindful of associated costs. Services like EC2 and S3 operate on a pay-as-you-go model, meaning expenses can accumulate rapidly depending on usage. Proper cost management tools, such as AWS Budgets and AWS Cost Explorer, should be used to avoid unexpected charges.

For those new to AWS, the following resources are available:

AWS Getting Started Guide: This guide provides tutorials and step-by-step instructions to help users familiarize themselves with AWS services and best practices.
AWS HealthOmics: A purpose-built service designed to help researchers store, query, and analyze genomic, transcriptomic, and other omics data efficiently.
Introduction to AWS for Bioinformatics: A practical guide for bioinformatics researchers looking to leverage AWS services for high-performance computing and data storage.

Important Warning

AWS is not recommended for beginners and should only be used by individuals with advanced experience in cloud computing and resource management. Improper use can result in unexpected high costs. Those seeking free or low-cost alternatives for cloud computing should consider platforms like JetStream2, which provides cloud-based computational resources for scientific research at no cost to educators and students.

By leveraging AWS's extensive suite of services, experienced users in genomics can build scalable and efficient workflows to handle the complexities of sequencing data analysis.

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Using Jupyter for Interactive Nanopore Analysis

Jupyter is an open-source tool designed for interactive computing and reproducible research It provides a flexible environment, including for analyzing Nanopore data, combining code, text, and visualizations in a single document. Educators and researchers can use Jupyter Notebooks or JupyterLab to conduct bioinformatics workflows, explore sequencing datasets, and develop tutorials that enhance student learning.

Benefits Jupyter

Jupyter Notebooks are widely used in computational biology because they allow users to create, share, and execute self-contained analytical workflows. Jupyter provides:

An interactive environment for processing sequencing data step by step.
Documentation alongside analysis, ensuring clarity in computational workflows.
Support for multiple programming languages, including Python, R, and Bash.
Integration with data visualization tools, allowing real-time plotting of sequencing results.
Improved collaboration and reproducibility, making it easier for students and researchers to share results and rerun analyses.

Installing Jupyter Notebook and JupyterLab

There are two main ways to install Jupyter: Jupyter Notebook and JupyterLab. Jupyter Notebook provides a simplified interface, while JupyterLab offers a more advanced environment with additional features.

Installation via Anaconda (Recommended)

Anaconda is the preferred installation method for most users, as it includes Jupyter and key scientific computing libraries.

Installation via pip

For those preferring a minimal installation, Jupyter can also be installed using pip, the Python package manager.

For detailed instructions, visit the Jupyter installation guide.

Google Colab: A Cloud-Based Alternative

Google Colab is an alternative to Jupyter that runs entirely in the cloud requiring no installation. It provides free access to computational resources and integrates with Google Drive for storing and sharing notebooks. Google Colab is particularly useful for classroom settings where students may not have access to high-performance local machines.

Key benefits of Google Colab include

No setup or installation required.
Free (but limited) access to GPUs.
Seamless integration with cloud storage for easy data access.
Built-in support for popular bioinformatics libraries.

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Ended: Cloud Computing and other Platforms

Ended: Bioinformatics

Teaching Resources ↵

Faculty Mentoring Networks: A Community of Practice for Educators

What is a faculty mentoring network?

A Faculty Mentoring Network (FMN) is a structured, collaborative group of educators working together to develop and implement innovative teaching strategies FMNs provide a supportive environment for faculty to share experiences, discuss challenges, and refine teaching approaches. In the context of Nanopore sequencing an FMN can help educators navigate the integration of real-world genomics research into their classrooms.

FMNs are an example of a Community of Practice (CoP)—a group of people who share a common concern or passion and learn how to improve their practice through regular interaction. CoPs are particularly valuable in education, where faculty may need to acquire new technical skills such as those required for bioinformatics and hands-on sequencing experiments.

Why Join a community of practice?

Educators new to Nanopore sequencing and/or bioinformatics may feel overwhelmed by the breadth of technical knowledge required. A Community of Practice, such as a Faculty Mentoring Network, provides:

Collaborative Learning – Educators can share lesson plans, sequencing protocols, and troubleshooting strategies.
Expert Guidance – Experienced mentors provide step-by-step support, making it easier to overcome technical barriers.
Peer Support – Networking with colleagues facing similar challenges fosters a sense of community and shared growth.
Classroom Implementation Strategies – Participants can discuss best practices for integrating sequencing and genomics into undergraduate education**.
Resource Sharing – Members often gain access to teaching materials, datasets, and computational tools developed by the group.

Additional Reading

Adams, S., Tesene, M., Gay, K., Brokos, M., McGuire, A., Rettler-Pagel, T., & Swindell, A. (2023). Communities of Practice in Higher Education: A Playbook for Centering Equity, Digital Learning, and Continuous Improvement. Every Learner Everywhere. https://www.everylearnereverywhere.org/resources/communities-of-practice-in-higher-education/.

The Nanopore Faculty Mentoring Network

The Nanopore FMN, hosted on QUBESHub, is designed to support educators who want to bring Nanopore sequencing into their classrooms**. Participants in this FMN will:

Gain hands-on experience with MinION sequencing and data analysis.
Work with mentors and peers to design classroom-friendly experiments
Develop teaching modules that can be adapted to different learning environments.
Explore strategies for data analysis, storage, and management in an educational setting.

For the latest updates on upcoming mentoring opportunities visit the **Nanopore Faculty Mentoring Network page. We also suggest joining the Slack discussion using this invitation link.

Joining a FMN is an excellent way to enhance professional development, collaborate with peers, and build confidence in bringing cutting-edge genomics into undergraduate and high school classrooms.

Course-Based Research Experiences (CUREs): Transforming STEM Education

What Are Course-Based Research Experiences (CUREs)?

Course-Based Undergraduate Research Experiences (CUREs) integrate authentic scientific research into undergraduate and high school courses, allowing students to engage in real-world inquiry as part of their coursework Unlike traditional laboratory exercises, which often follow a predetermined set of instructions to verify known scientific concepts, CUREs immerse students in open-ended investigations enabling them to contribute to the broader scientific community.

According to SERC at Carleton College, CUREs have five defining characteristics:

Use of Scientific Practices – Students formulate hypotheses, design experiments, collect and analyze data, and interpret results.
Discovery of New Knowledge – Unlike conventional labs, which confirm established facts, CUREs contribute novel insights to a research field.
Broadly Relevant Work – Research projects address unanswered questions that have significance beyond the classroom, often contributing to published research or larger scientific initiatives.
Collaboration – Students work in teams to share ideas, troubleshoot problems, and collectively advance the research.
Iteration – Students revise their approaches based on their findings, developing resilience and problem-solving skills as they refine their research methods.

Why Are CUREs Important?

Integrating research into courses enhances STEM education in several key ways:

Increases Student Engagement – Conducting original research is more stimulating than routine lab exercises, leading to higher student motivation
Improves Retention in STEM – Studies indicate that students involved in research are more likely to persist in STEM majors and careers.
Fosters Critical Thinking and Scientific Literacy – Engaging in research helps students develop skills in data analysis, communication, and problem-solving.
Expands Access to Research – CUREs enable more students, especially those at minority-serving institutions (MSIs) or under-resourced schools to participate in research experiences that they might not otherwise access.

Nanopore Sequencing and CUREs

Nanopore sequencing is well-suited for CUREs in biology and genomics because it allows students to generate and analyze real DNA sequence data within a classroom setting. By incorporating DNA barcoding, metagenomics, or genome sequencing, educators can design research projects that help students:

Address authentic scientific questions, such as identifying environmental microbes or sequencing unknown species.
Use cutting-edge sequencing technologies, mirroring real-world research settings.
Develop bioinformatics and data analysis skills, preparing them for careers in STEM fields.

CUREs that incorporate Nanopore sequencing provide high-impact, interdisciplinary learning experiences exposing students to molecular biology, computational biology, and scientific inquiry.

Additional Reading

For more information on CUREs and their impact, visit the [CUREnet website](https://serc.carleton.edu/curenet/whatis.html.
A Guide to Course-based Undergraduate Research: Developing and Implementing CUREs in the Natural Sciences. First Edition Edition. ISBN-13: 978-1319367183. Publisher website.
AAAS (American Association for the Advancement of Science) (ed. C Brewer & D. Smith). (2011). Vision and Change in Undergraduate Biology Education: A Call to Action. ISBN#: 978-0-87168-741-8. https://visionandchange.org/finalreport/.
Auchincloss LC, Laursen SL, Branchaw JL, Eagan K, Graham M, Hanauer DI, Lawrie G McLinn CM, Pelaez N, Rowland S, Towns M, Trautmann NM, Varma-Nelson P, Weston TJ, Dolan EL. (2017). Assessment of course-based undergraduate research experiences: a meeting report. CBE Life Sci Educ. 13(1):29–40 doi: 10.1187/cbe.14-01-0004.
Dolan EL. (2016) Course-based Undergraduate Research Experiences: Current Knowledge and Future Directions. Paper commissioned for the Committee on Strengthening Research Experiences for Undergraduate STEM Students, Board on Science Education, Division of Behavioral and Social Sciences and Education, Board on Life Sciences, Division of Earth and Life Studies. Washington, D.C.: National Academies of Sciences, Engineering and Medicine. Available https://sites.nationalacademies.org/cs/groups/dbassesite/documents/webpage/dbasse_177288.pdf.

Teaching Resources

This page provides educators with a collection of instructional materials designed to support the integration of Nanopore sequencing into classroom and laboratory settings. Whether you are introducing DNA sequencing concepts, guiding students through hands-on sequencing experiments, or developing bioinformatics skills, these resources will help facilitate engaging and effective teaching.

How to Contribute

We welcome contributions from educators! If you have developed lesson plans, presentations, assessments, or other teaching materials, please consider sharing To contribute, contact williams@cshl.edu.

Slide Presentations

This section includes downloadable slides that educators can use for lectures and classroom discussions.

Presentation: Nanopore Introduction Slides
- Authors: Holly Nance, Jennifer Katcher, Barbara Murdoch, Elizabeth Hasenmyer, Olga Kopp, Cecilia Noecker, Nauapaka Zimmerman, Margaret Young, Val Carson.
Presentation: Long-Read Sequencing Technology
- Author: Katie M. Sandlin
- Affiliation: Genomics Education Partnership
Presentation: Example of NC State Undergrad/Grad Course
- Author: Carlos C. Goller & Pricilla Rozario
- Affiliation: North Carolina State University

Videos

Here, you will find educational videos that explain key concepts and demonstrate laboratory techniques.

Assessments

To gauge student understanding, we provide quizzes, problem sets, and concept questions.

Comming soon.

Collections, curricular Plans, handouts, and Materials

For educators designing course modules or research-based experiences.

Fulfilling the Promise of Nanopore Sequencing in Education
Author: The attendees of the September 2024 Banbury Meeting
- Description: This website contains more than 20 abstracts and presentations relevant to Nanopore sequencing in education
Website: Fulfilling the Promise of Nanopore Sequencing in Education - The Banbury Center Cold Spring Harbor Laboratory September 21–24, 2024

Meeting Summary:

Nanopore sequencing technology offers inexpensive, real-time analysis of individual DNA molecules—potentially making DNA sequencing available anytime, anyplace, to anyone. This technology holds particular promise in bioscience education, where we envision a miniature Nanopore sequencer in every teaching lab, with students at all levels generating and exploring meaningful data.

For these reasons, we held a small meeting, “Fulfilling the Promise of Nanopore Sequencing in Education,” on September 21–24 at the Banbury Center of Cold Spring Harbor Laboratory. Against this backdrop, 25 scientists and educators met at Cold Spring Harbor Laboratory’s Banbury Center in September to discuss how to fulfill this promise of nanopore sequencing in education. During the meeting, we learned of large existing audiences of students who could readily use nanopore sequencing for projects on DNA barcoding (popularized by the DNALC ), bacteriophage analysis (popularized by the Howard Hughes Medical Institute), and identification of antibiotic-producing bacteria (popularized by the University of Wisconsin). We also heard great examples of nanopore class projects – from assessing microbial diversity in local waterways to cataloging plants visited by bees, to sequencing daffodil chloroplast genomes.

Attendees joined break out groups to discuss the promises and challenges of Nanopore sequencing, as well as proposals for broad implementation. Key themes included sustained training, resource accessibility, and community collaboration. Continuous mentorship, including virtual teaching assistants and "train-the-trainer" programs were seen as vital for long-term success. Participants highlighted the importance of creating user-friendly kits and clear protocols to make genomic research accessible across educational levels. Partnerships among educational institutions, industry, and community organizations are crucial for sharing resources and fostering a genomics-literate workforce. The DNALC, which signed an MOU with Oxford Nanopore last year, will continue working with the organizations represented at the meeting as we lay groundwork for popularizing this technology into the classroom.

This meeting is supported by grants from the National Science Foundation: Improving Undergraduate STEM Education (#1821657), and Advanced Technological Education (#1901984). Additional travel support is provided by Oxford Nanopore Technologies.

Organized by:
- Anna Feitzinger, Cold Spring Harbor Laboratory DNA Learning Center
- Dave Micklos, Cold Spring Harbor Laboratory DNA Learning Center
- Jonathan Pugh, Oxford Nanopore Technologies
- Jason Williams, Cold Spring Harbor Laboratory DNA Learning Center
Collection: 16S Barcoding microbiome study

Author: Hui-Min Chung, University of Western Florida
- Description: Files based on materials shared with TAs when preparing the microbiome project in my genetics lab when we prepared the microbiome project.
  - Application of Nanopore seq tech in Undergraduate Research: Walk through–16S barcoding kit
    - Catagory: Presentation
    - File: Download
  - Application of Nanopore seq tech in microbiome study: 16S Barcoding project
    - Catagory: Presentation
    - File: Download
  - 16S Barcoding Library Preperation
    - Catagory: Lab Handout
    - File: Download

Key Papers

This section includes foundational and recent research papers on Nanopore sequencing and its applications in education and research. These papers provide background on the technology, its capabilities, and best practices for implementation in the classroom.

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Educator Profiles

If you've done one or more rounds of teaching Nanopore at the secondary or undergraduate level, please share your profile to highlight your work and connect with this growing community!

Karen Barnard-Kubow

: James Madison University

: Harrisonburg, Virginia, United States

: Undergraduates

: email

: plants (chloroplast genome sequencing), also some 16s metabarcoding

: Experienced with Nanopore

About Karen

Classroom research question(s)

We run a module in our core Genetics lab (10 lab sections of 24 students) asking if structural variation in the chloroplast genome of Campanula americana is related to strength of cytonuclear genetic incompatibility. The focus of the 2-week module is to introduce students to the concepts of speciation, reproductive isolation, and genetic incompatibility, as well as chloroplast function and structure, and long-read next generation sequencing, with a focus on Nanopore sequencing.

Using Nanopore in the classroom

The module utilizes the plant species, Campanula americana, which exhibits cytonuclear incompatibility in the form of hybrid seedlings exhibiting chlorosis (reduced chlorophyll production) leading to yellow or white seedlings. The week before the module starts each group plants hybrid seeds from a different cross, which they will score for strength of incompatibility once the plants germinate by scoring their phenotype (green=no incompatibility, yellow=moderate incompatibility, while=strong incompatibility). We start the first week with an introductory presentation about the project and Minion sequencing. The students then interact with used flow cells, where each student practices priming and loading a used flow cell with water. The students then do a trial run-through of the bioinformatic analysis pipeline which is carried out in Google Colab, so all students can run the pipeline on their own computers. The following week, students use the bioinformatics pipeline to analyze Minion sequence data and assemble the chloroplast genome of one of the parents of their seedlings. In the pipeline they check the quality of the reads using Nanoplot, assemble the chloroplast genome using Flye, visualize the assembly using Bandage, and annotate it using GeSeq. They then get the genome assembly for the second parent of their hybrid seeds from another group and align the two assemblies and score structural variation between the two chloroplast genomes using Assemblytics. Then we combine data across all eight groups and make a graph with number of structural variants on the x and strength of genetic incompatibility on the y, to ask if there is a relationship between extent of structural variation between chloroplast genomes and strength of genetic incompatibility.

URLs

https://github.com/kbkubow/NanoporePlastidAssemblyModule/tree/main
Yokshitha Bathula

: HudsonAlpha Institute for Biotechnology

: Huntsville, Alabama, United States

: High school

: email

: eDNA

: Some experience with Nanopore

About Yokshitha

Educational Goal

Exploring bacterial biodiversity and antibiotic resistance across different soil ecosystems.

Using Nanopore in the classroom

We use Nanopore sequencing as part of a semester-long independent project with high school students. The students extract eDNA from soil samples and perform 16S sequencing. They use the results from the EPI2ME workflow to do a comparative study of bacterial diversity and look for antibiotic-resistance genes that align with the microbial lab results.
- Anna Feitzinger
  
  : Cold Spring Harbor Laboratory, DNA Learning Center
  
  : Brooklyn, New York, United States
  
  : Support for all educators, High school, Undergraduates
  
  : email
  
  : plants, bacteria, eDNA, human
  
  : Experienced with Nanopore
  
  About Name
  
  Educational Goal
  
  One research question we ask is how does human activity impact the local waterways of NYC?
  
  Using Nanopore in the classroom
  
  I teach a semester long college course in which students explore the biodiversity and water quality of local waterways in New York City with a focus on eDNA. In addition, I work with high school students, introducing them to genomics and bioinformatics during summer workshops.
  
  URLs
  - DNA Learning Center
Bob Kuhn

: FCS Innovation Academy STEM High School

: Georgia, United States

: High school

: Reach me on Slack

: 16S DNA Barcoding, Phage Genomes

: Getting started with Nanopore

About Bob

Classroom research question(s)

How do bacterial abundance and assemblages change across environments and scales?

Using Nanopore in the classroom

We have used Nanopore 16S barcoding to investigate simple variable differences such as bean beetles dosed with probiotics, salamander species skin swabs, different vegetables in kimchi, and bacterial profiles in Georgia lakes. We also hope to begin bacteriophage genome sequencing in fall 2025 as part of our phage discovery program.

URLs

https://fcsinnovationacademy.fultonschools.org/
Sylvia Franke McDevitt

: Skidmore College

: Saratoga Springs, NY, United States

: Undergraduates

: email

: 16S barcoding

: Some experience using Nanopore

About Sylvia

Educational Goal

We are analyzing microbial communities from a variety of sources to answer questions like changes in the microbiome of mice, impact of heavy metal contamination in soil, or bacteria in sourdough.

Using Nanopore in the classroom

In my research we are looking at the microbial communities of creek sediments near old manufacturing sites, which have historically released heavy metals. Additionally we are analyzing fecal samples of mice with and without zinc supplement. The latter project is being done both in my research group as well as in my upper-level class. In my intermediate level General Microbiology students are proposing their own research question comparing microbial communities using the 16S Barcoding workflow.
James Melton

: Spelman College

: Atlanta, Georgia, United States

: Undergraduates

: email

: Bacteriophage

: Experienced with Nanopore

About James

Educational Goal

This research is conducted as a part of the SEA-PHAGES (Science Education Alliance- Phage Hunters Advancing Genomics and Evolutionary Sciences) program to isolate and characterize phages from soil.

Using Nanopore in the classroom

In the classroom, we perform whole genome sequencing of Arthrobacter globiformis and Gordonia rubripertincta phages. Approximately ten students isolate a phage from soil and gain hands-on experience with Nanopore sequencing and bioinformatics.

URLs
- James Melton
Fernando Nieto

: State University of New York, Old Westbury

: New York, United States

: High school, Undergraduates

: email

: Bacteriophages, and 16S/18S metabarcoding.

: Some experience with Nanopore

About Fernando

Educational Goal

We are interested in understanding microbial diversity and microbiome change patterns in the environment and in humans.

Using Nanopore in the classroom

We are using Nanopore sequencing for sequencing bacteriophage genomes isolated by students from soil samples as part of the SEA-PHAGES program. We are also trying for the first time to use ONT for 16S and 18S sequencing to analyze environmental and human oral microbiome. These activities are integrated as CUREs into our undergraduate biology courses that are part of the major, i.e. ecology and microbial ecology. HS students are also involved as part of of their participation in the Science Technology Entry Program (STEP) funded by NYSED.

URLs
- Fernando Nieto
Robyn P

: Bridgewater College

: Virginia, United States

: Undergraduates

: email

: amphibians

: Getting started with Nanopore

About Robyn

Educational Goal

Looking at vernal pool use by amphibians breeding in Virginia.

Using Nanopore in the classroom

I am hoping my department will purchase an MinIonMk1D unit. I am working with the microbiologist on 16S sequences from environmental samples, and I am interested in metagenomic samples of eDNA from vernal pools.
Jonathan Pugh

: Oxford Nanopore Technologies

: Oxford, United Kingdom

: Support for all educators

: email

: Experienced with Nanopore

About Jon

Educational Goal

I facilitate educators to do more of their great work, and collaborate with key organisations to utilise existing networks of educators

Using Nanopore in the classroom

Here to facilitate, collaborate, champion, and protect!

URLs
- https://nanoporetech.com/about/education
Publications
- The Current State of Nanopore Sequencing
- Oxford Nanopore Technologies - nanopore sequencing education
Jason Williams

: Cold Spring Harbor Laboratory, DNA Learning Center

: Cold Spring Harbor, New York, United States

: Support for all educators, High school, Undergraduates

: email

: Plant and microbial DNA, DNA Barcoding

: Experienced with Nanopore

About Jason

Educational Goal

Develop resources that allow faculty to bring Nanopore into the classroom.

Using Nanopore in the classroom

As the lead PI for the project, Jason was the first to introduce Nanopore sequencing at the DNA Learning Center in 2014 as a potential activity for our students. Since then, it has been a long journey of technological and curricular development to bring this technology into the hands of educators across the country and around the world.

URLs
- DNA Learning Center
Publications
- This eBook!
Miguel Urdaneta

: University of Puerto Rico, Río Piedras Campus

: San Juan, Puerto Rico, United States

: Undergraduates

: email

: Insects: Diaprepes abbreviatus, Apis mellifera / DNA Barcoding / 16S / Ligation-based Amplicons

: Experienced with Nanopore

About Miguel

Educational Goal

Antibiotic-resistance patterns in gut-microbes of insects and their potential role as probiotics and regulators of the gut-brain axis. Potential pathogens variations on Puerto Rico honey bees Apis mellifera.

Using Nanopore in the classroom

In our undergraduate Applied Microbiology laboratory, we have been used the Nanopore 16S Barcoding and the Ligation-based Amplicon Barcoding protocols to investigate the bacterial diversity on the Caribbean pest insect Diaprepes abbreviatus and in the honeybees Apis mellifera.

Ended: Teaching Resources

Other Resources ↵

Frequently Asked Questions (FAQ) for New Nanopore Users

This FAQ addresses common questions educators and first-time users may have about Nanopore sequencing, its applications, and how to integrate it into the classroom.

Getting Started

Q: What is Nanopore sequencing?
A: Nanopore sequencing is a method of DNA (RNA and perhaps one day protein) sequencing that detects changes in electrical current as nucleic acids pass through a biological nanopore. This allows for real-time sequencing of long DNA fragments.

Q: What equipment do I need to get started with Nanopore sequencing?
A: The MinION device is the most accessible option, along with a computer that meets system requirements, a flow cell, sequencing kits, and basic molecular biology equipment.

Q: How much does it cost to start sequencing?
A: Costs vary depending on the setup. A MinION starter pack (device + flow cells + sequencing kit) is around $2,000, but ongoing costs include consumables like flow cells and reagents. See our time and costs guide.

Q: Do I need prior experience in sequencing or bioinformatics to use Nanopore sequencing?
A: No, but familiarity with basic molecular biology techniques and an understanding of sequencing principles will help. The platform provides user-friendly software such as MinKNOW and EPI2ME. See also software guide.

Q: How does Nanopore sequencing compare to other sequencing methods?
A: Unlike Illumina sequencing, which provides short, highly accurate reads, Nanopore sequencing produces long reads that can span structural variants and repetitive regions, with real-time data generation.

Using Nanopore in the Classroom

Q: How can I integrate Nanopore sequencing into my curriculum?
A: Start with simple experiments, such as microbial identification via 16S sequencing, and progressively introduce DNA barcoding or small genome sequencing.

Q: Is it safe for students to handle Nanopore sequencing experiments?
A: Yes, with appropriate safety precautions. Many classroom protocols use non-hazardous DNA extraction methods and minimize toxic reagents.

Q: What type of experiments can students do with Nanopore sequencing?
A: Common classroom experiments include: - Microbiome studies (16S sequencing) - DNA barcoding (identifying species using specific gene markers) - Small genome sequencing (e.g., bacteriophage or bacterial genomes)

Q: How long does a typical sequencing experiment take?
A: From sample preparation to sequencing: - DNA extraction: 1-2 hours - Library preparation: 1-2 hours - Sequencing: 1-24 hours (depending on read depth) - Data analysis: 1-3 hours These are general answers, check your specific protocol for more details.

Q: Do students need their own sequencing devices?
A: No, a single MinION device can be shared, with students processing different samples in barcoded multiplexed runs.

Sequencing and Data Handling

Q: What kind of samples can be sequenced?
A: Almost any biological sample containing DNA, including: - Environmental samples (soil, water, surfaces) - Microbial cultures - Human samples (with appropriate ethics approval) - Plant or animal tissues

Q: How much DNA is required for sequencing?
A: This depends on the kit:

Rapid barcoding kit: ~10 ng per sample
Ligation sequencing kit: ~100 ng per sample
16S sequencing kit: ~10 ng per sample

Q: How is sequencing data stored and analyzed?
A: Sequencing generates POD5 files (raw signal data) and FASTQ files (nucleotide sequences). Data can be analyzed using EPI2ME, Galaxy. See also data guide.

Q: What storage is needed for sequencing data?
A: A minimum of 500GB storage is recommended, with 1TB preferred. Cloud-based solutions like CyVerse, JetStream2, or AWS S3 are also options. See also data guide.

Q: Can Nanopore sequencing be used for metagenomics?
A: Yes, Nanopore sequencing can be used for metagenomic studies, enabling long-read assembly of complex microbial communities.

Troubleshooting and Maintenance

Q: What if my sequencing run fails?
A: Common issues include:

Low DNA input → Ensure enough high-quality DNA is used.
Flow cell clogging → Use a flush kit to unclog pores.
Low sequencing yield → Check sample preparation and quality.

Q: How should flow cells be stored?
A: Flow cells should be kept at 2-8°C and used before the expiration date. If not used immediately, they should not be frozen.

Q: How do I clean and reuse a flow cell?
A: MinION Flow cells can be washed and reused 1-2 times. For another sequencing run you must use a Flow Cell Wash Kit. Flongle flow cells are one-time use.

Q: How long does a flow cell last?
A: A MinION flow cell typically lasts for 1-2 sequencing runs with an 11-week shelf life. Flongles typically last 4-6 weeks in our experience.

Advanced Topics

Q: Can I run multiple samples at once?
A: Yes, multiplexing allows you to sequence multiple barcoded samples in the same run, reducing costs.

Q: Can I sequence RNA instead of DNA?
A: Yes, direct RNA sequencing is possible with Nanopore, allowing you to study full-length transcripts and modifications. As of yet, we don't cover this topic as the specialized requirements for working with RNA is likely less common in general classroom settings.

Q: Can I use cloud computing for sequencing analysis?

A: Yes, JetStream2, AWS, and EPI2ME offer cloud-based solutions for basecalling and data analysis.

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Nanopore Sequencing Glossary

This glossary provides definitions of key terms related to Nanopore sequencing, offering educators and first-time users a foundational understanding of the technology and its associated concepts.

A

Adapter
Short DNA or RNA sequences ligated to sample fragments to facilitate their attachment to sequencing platforms.

Alignment
The process of arranging sequences to identify regions of similarity, which may indicate functional, structural, or evolutionary relationships.

Amplicon
A DNA fragment that has been amplified by PCR.

B

Base Modification
Chemical alterations to DNA bases, such as methylation, which can affect gene expression.

Basecalling
The process of determining the sequence of nucleotides (bases) from raw signal data generated by the sequencing platform.

Barcode
Short, unique sequences added to DNA fragments to identify and differentiate multiple samples sequenced together. See also Indexing.

C

Chimera
A sequence artifact formed by the joining of two or more unrelated DNA fragments.

Consensus Sequence
A sequence derived from the alignment of multiple sequences, representing the most common nucleotide at each position.

Contig
A contiguous sequence assembled from overlapping reads.

Coverage
The number of times a nucleotide is read during sequencing; higher coverage increases confidence in the accuracy of the sequence.

D

De Novo Sequencing
Sequencing a novel genome without a reference, assembling the sequence from scratch.

Demultiplexing
The process of sorting sequences from a mixed sample pool into individual datasets based on their barcodes.

Direct RNA Sequencing
Sequencing RNA molecules directly without converting them to cDNA, preserving base modifications.

DNA Library
A collection of DNA fragments prepared for sequencing.

Dorado
A basecalling software developed by Oxford Nanopore Technologies for translating raw nanopore signals into nucleotide sequences.

E

Epigenetics
The study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence.

Epigenome
The complete set of epigenetic modifications on the genetic material of a cell.

F

FAST5
A file format previously used by ONT to store raw signal data from nanopore sequencing. It has been replaced by POD5.

FASTQ
A common file format for storing nucleotide sequences and associated quality scores.

Flow Cell
A consumable device containing nanopores through which nucleic acids pass during sequencing.

G

H

High Molecular Weight (HMW) DNA
Long DNA fragments essential for obtaining long reads in sequencing.

I

Indexing
The use of unique sequences (indexes), also called barcodes, to tag DNA fragments from different samples, enabling their identification after pooled sequencing.

L

Library Preparation
The process of preparing DNA or RNA samples for sequencing, including fragmentation, adapter ligation, and amplification.

Ligation
The enzymatic process of joining two DNA fragments together.

Long Reads
Sequences that are typically longer than 10,000 base pairs, advantageous for resolving repetitive regions and structural variants.

M

MinION
A portable nanopore sequencing device developed by Oxford Nanopore Technologies.

Rapid library preparation kits

Multiplexing
Sequencing multiple samples simultaneously by using unique barcodes for each sample.

N

Nanopore
A nanometer-scale pore used in sequencing to detect changes in ionic current as nucleic acids pass through, allowing base identification.

Native library preparation kits A library preparation kit that uses ligation to attach sequencing adapters; is longer and uses additional 3rd party reagents compared to the rapid library preparation kits but also results in longer fragment lengths.

Oxford Nanopore Technologies (ONT)
The company that developed and commercialized nanopore sequencing technology.

P

Pore
A tiny opening in a membrane through which molecules can pass.

POD5
The current file format used by ONT to store raw sequencing signal data.

PromethION
A high-throughput nanopore sequencing device designed for large-scale projects.

Q

Quality Score (Phred Score)
A metric that indicates the confidence of a base call; higher scores represent greater confidence.

R

Rapid Adapter
Short DNA sequence ligated to sample fragments to facilitate their translocation through the nanopore.

Rapid library preparation kits A library preparation kit that uses a transposase complex to attach sequencing adapters; is quicker and uses fewer 3rd party reagents than the native library preparation kit but also sheers the DNA resulting in smaller fragment lengths.

Read
A sequence of nucleotides generated by a sequencing machine from a single DNA or RNA fragment.

Real-Time Sequencing
The ability to analyze sequencing data as it is generated, enabling immediate insights.

Reference Genome
A representative example of a species' genome used as a standard for comparison.

S

Single Molecule Sequencing
Sequencing technologies that read individual DNA or RNA molecules without amplification.

T

Throughput
The amount of data generated by a sequencing platform in a given time frame.

Translocation
The movement of nucleic acid molecules through a nanopore during sequencing.

U

Ultra-Long Reads
Sequences exceeding 100,000 base pairs, useful for assembling complex genomes.

V

Variant Calling
The process of identifying variations (e.g., SNPs, insertions, deletions) between sequences and a reference genome.

Y

Yield
The total amount of data (in bases) produced by a sequencing run.

Z

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Ended: Other Resources

Contribute ↵

Contributions

This e-book is intended to be a collaborative resource developed by and for the community of educators using Nanopore sequencing in their classrooms and laboratories. As more instructors integrate real-world DNA sequencing experiences into their courses, sharing materials, insights, and best practices helps build a stronger foundation for effective, accessible, and innovative teaching.

We welcome contributions from educators, researchers, and practitioners to expand and refine this resource. There are many ways to contribute, ranging from submitting instructional materials to providing feedback on existing content.

Ways to Contribute

Provide feedback

If you've used materials from this e-book and found areas for improvement, or if you'd like to suggest new topics, your feedback is valuable. Educators who are actively using Nanopore sequencing with students can help ensure that our content remains:

Practical – Aligned with real-world classroom and laboratory settings
Accurate – Updated with the latest scientific knowledge and best practices
Inclusive – Designed to be accessible to a diverse range of students

To share feedback options include:

Email williams@cshl.edu
Participate in the discussion at QUBES Nanopore Network or Nanopore Network Slack.
Visit the website's GitHub page and submit an issue.

On pages that may be frequently updated, you will also see a feed from our Slack channel.

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If you have developed lesson plans, presentations, worksheets, videos, or assessments, we encourage you to share them with the community. Contributions may include:

Lecture slides or presentations
Step-by-step protocols for DNA extraction, sequencing, or data analysis
Sample datasets for classroom use
Assessments or concept questions to test student understanding
Complete course modules or research experiences (CUREs)

Submit research and case studies

If you have conducted research on Nanopore sequencing in the classroom or developed a case study about how it has impacted student learning, we encourage you to share your findings. Potential contributions include:

Published papers or preprints on Nanopore-based education initiatives
Case studies of student-led sequencing projects
Institutional reports on course-based research experiences (CUREs

Contribute to the glossary or FAQs

The glossary and FAQ sections are designed to help newcomers quickly understand technical terms and common questions about Nanopore sequencing. If you notice missing terms or important questions that should be addressed, your contributions can enhance clarity for future educators and students.

Contribution templates

You can visit examples/protocol-template.md to see and download a template for contributing protocols.

Editing the website

The full authoring workflow—the process of building this entire site—relies on two GitHub repositories, an a variety of publishing tools, the most important of which are MkDocs and Material for MkDocs. You will need a bit of GitHub knowledge and Command line knowledge to fully manipulate these tools.

Overview

In the MkDocs system your 'docs' are a collection of markdown files; every page in this site is built as a markdown file. There is also a mkdocs.yml file which contains important metadata about how the site is organize (e.g., the title of the site, the navigation bar, etc.).

Once you have made a page(s) in markdown, there is some serve command you can use to serve the site (e.g. mkdocs serve) to build an html version of the site as a preview you can browse locally. When you are ready, there will also be some deploy command which pushes the html site to GitHub to be served as a website.

This site, is a bit more complicated.

The problem, is that in the build process, the unrendered markdown docs remain on my (the author's) computer, and what is pushed to github are only the HTML files organized however MkDocs has decided. We don't want people editing the HTML files—it's difficult to do, and the point is that editing should take place in markdown only. If I want other people to be able to edit my original markdown files they can't. Therefore, this site is split into two repositories:

nanopore_e-book-dev: Is a private repository (i.e., you must request access) where the unrendered docs live.
ebook-website: Is a public repository where the built site lives. If you check that site you will see that it basically containes HTML files.

The authoring workflow is something like:

Authoring steps

Fork and clone repository and install software dependencies

If needed, contact williams@cshl.edu or feitzin@cshl.edu to be added to the repository as a collaborator; you will need to share your GitHub ID.
On github, go to nanopore_e-book-dev and create a fork to your own GitHub account.
Clone the nanopore_e-book-dev repository.
```
git clone YOUR FORKED REPOSITORY
```
Use python pip to install all of the software dependencies in the requirements.txt; verify that all packages have installed successfully.
```
pip install -r requirements.txt
```
or
```
pip3 install -r requirements.txt
```

Edit the website in markdown form and view a local preview

Change into the nanopore_e-book-dev directory you cloned.
```
cd nanopore_e-book-dev
```
1. Optional, create a branch if desired.
Use a text editor (e.g., atom) to make changes to the documentation.
Tip

We are using the Material theme. Explore the site's documentation for extensive examples on how to use:
- admonitions (e.g., tip boxes)
- annotations
- navigation
Use mkdocs serve to generate a local preview of your site. This will be a 'live' preview which will refresh in your browser everytime you save a change to your website.
```
mkdocs serve
```
Copy the URL (e.g. 'http://127.0.0.1:8000/nanoporenetwork/ebook-website/') to a web browser to preview. In the terminal use CNTRL+C to stop serving the preview site.

Commit your changes, push, and create a pull request

Add all changed files and commit your change

git add --all

git commit -am "commit message"

Push your change to your fork on GitHub
```
git push
```
Create a pull request on Github. The website maintainers will review your changes, suggest edits, and/or incorporate your requested changes!

Making changes to the live site

Edits and changes are done as above. In principle, the live site will have versioned releases using mike.

Create a version of the site using mike; the command string will contain a version and, optionally, and alias (e.g. mike deploy [version] [alias]...)
```
mike deploy February-2025-alpha latest
```
Tip

Versioning

Versioning for this website shall be:
- Major Versions: Month-YYYY (e.g., March-2025); for major releases (e.g., May, August, December)
- Minor Versions: Month-YYYY-a (e.g. March-2025-a, March-2025-b, ...); for critical updates between major releases.
Typos, broken links, etc. can be fixed any time without a new release. Correcting outdated information, adding new links to external documents, etc. should trigger a minor version. Substantially new content should be done at major version releases.

Set the version to default

mike set-default February-2025-alpha # or whatever version

Build the site and preview. Copy the URL (e.g. 'http://localhost:8000/') to a web browser to preview. In the terminal use CNTRL+C to stop serving the preview site. The version dropdown should appear in the site header.
```
mike serve
```
Deploy the site from the development repository to the ebook website depository. Note, you will only be able to do this if you also have write access as a collaborator to the main site.
```
mike set-default --push latest -r https://github.com/nanoporenetwork/ebook-website.git --allow-empty
```

Creating formal releases and updating website citation

Following these instructions for logging into a Zenodo account and adding the main repository. When you create a release you will get an update to the Zenodo record.

✓	Ingredient	Volume
	200 fmol Amplicon DNA	11.5 µL
	Diluted DNA Control Sample (DCS)	1 µL
	Ultra II End-prep Reaction Buffer	1.75 μL
	Ultra II End-prep Enzyme Mix	0.75 μL

✓	Ingredient	Volume
	Nuclease free water	3 µL
	End-prepared DNA	0.75 µL
	Individual Native Barcode	1.25 μL
	Blunt/TA Ligase Master Mix	5 μL

✓	Ingredient	Volume
	Pooled barcoded sample	30 µL
	Native Adapter (NA)	5 µL
	NEBNext Quick Ligation Reaction (5X)	10 µL
	Quick T4 DNA Ligase	5 µL

Reagent	Volume
10 ng input DNA (from previous step)	15μl
LongAmp Hot Start Taq 2X Master Mix	25μl
Total	40μl

Reagent	Volume
Rapid Adapter (RA)	1.5μl
Adapter Buffer (ADB)	3.5μl
Total	5μl

Reagents	Volume
Sequencing Buffer (SB)	15 µl
Library Beads (LIB) (mixed immediately before use) or Library Solution (LIS) (if using)	10 µl
DNA Library	5 µl
Total	30 µl

Reagent	Volume
End-prepped DNA	7.5 µl
Native Barcode (NB01-24)	2.5 µl
Blunt/TA Ligase Master Mix	10 µl
Total	20 µl

Fragment Library Length	Flow Cell Loading Amount
Very short (<1 kb)	100 fmol
Short (1-10 kb)	35–50 fmol
Long (>10 kb)	300 ng

Reagent	Volume per Flow Cell
Flow Cell Flush (FCF)	1,170 µl
Bovine Serum Albumin (BSA) at 50 mg/ml	5 µl
Flow Cell Tether (FCT)	30 µl
Total Volume	1,205 µl

Nanopore for Educators eBook

Welcome

Why a guide for educators?

What this guide is not

Nanopore Network

Ask a question

Citation and licensing

Funding acknowledgment, and declared interests

Get Started ↵

Getting Started with Nanopore Sequencing

Why Nanopore sequencing is promising for education

What is Oxford Nanopore sequencing?

Am I ready to bring Nanopore sequencing into my classroom?

1. Technical readiness

2. Training and expertise

3. Budget and resources

4. Curriculum alignment

5. Student readiness

6. Community and support

7. Ethical and inclusive practices

What do my scores mean?

From absolute beginner to competent and confident

What this site offers

A journey of growth

Ten Steps to Getting Started with Nanopore

Essential steps for getting started with Nanopore sequencing

Estimating time and costs to get started with Nanopore

Example prep and teaching activities and timings

Example costs - Nanopore equipment

Example costs - Nanopore reagents and consumables

16S Kit (6 x 24 samples) - Microbial diversity

Rapid Barcoding Kit (6 x 24 samples) - DNA barcoding and small genomes

Ligation Kit (6 samples) - Small genomes

Start-up Checklists

Equipment, reagents, supplies, and computer technologies lists

Personal Protective Equipment

Molecular Biology Equipment

Nanopore Sequencing Equipment

Nanopore Sequencing Kits

Molecular Biology Consumables

Molecular Biology Reagents

Computers, accessories, software, and online accounts

Ended: Get Started

Equipment and Lab Management ↵

Oxford Nanopore Sequencing Hardware

The MinION device

MinION device models: Mk1B, Mk1C, and Mk1D

Flow cells

Other Nanopore sequencing platforms

Library Preparation and Sequencing Kits

Core Nanopore sequencing kits

16S Barcoding Kit 24 V14

Rapid Barcoding Kit 24 V14

Ligation Sequencing Kit V14

Additional sequencing reagents

Computer and Software Management for Nanopore Sequencing

Computer Requirements

Minimum System Requirements

Recommended Systems

Data Storage in Nanopore Sequencing Workflows

Software Requirements

MinKNOW

EPI2ME

Ended: Equipment and Lab Management

Laboratory Protocols ↵

Sample Collection ↵

DNA Sampling and Extraction for Nanopore Sequencing

Introduction

Basic principles of DNA extraction

DNA extraction recommendations by sample type and analysis endpoint

Phage

Bacteria

General microbial

Soil, environmental

Water, environmental

Plant

Insect

General

Ended: Sample Collection

Other Sequencing Protocols ↵