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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