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Annotated Experiment: 16S Sequencing

Examining Microbial Diversity — Nanopore "Shoe-ome" Sequencing

Protocol information

Protocol Credits

DNA sample source

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

Nanopore Sequencing

Computer and Bioinformatics

Reagents

Personal protective equipment

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

Sample collection and prep

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.

  1. 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
  1. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis: paper
  2. Performing accurate species-level bacterial identification with nanopore sequencing: paper
  3. 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.

Instruction tip

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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.

References

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

Part I - Preparing swab sample

  1. 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.

  2. Incubate the swab collection tube at 65˚C for 5 minutes.

  3. Carefully remove the swab from the collection tube.

  4. Label a microcentrifugue tube and transfer up to 1 mL of the preserved sample to the microcentrifuge tube.

  5. 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.

  6. Label a new microcentrifuge tube and carefully transfer 700 μL of supernatant, without the white layer debris, to the microcentrifuge tube.

  7. Add 100 μL of Binding Buffer I, mix by inverting the tube a few times, and incubate for 10 minutes on ice.

  8. Spin the lysate for 2 minutes at 20,000 x g (~14,000 RPM) to pellet any cell debris.

  9. 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.

  10. 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

  1. Assemble a spin column with one of the provided collection tubes.

  2. 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).

  3. Repeat step 2 with the remaining volume of lysate mixture.

Part III - wash column

  1. 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.

  2. Discard the flowthrough and reassemble the spin column with its collection tube.

  3. Apply 500 μL of Wash Solution A to the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM).

  4. Discard the flowthrough and reassemble the spin column with its collection tube.

  5. Repeat steps 3 and 4.

  6. 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

  1. Place the column into a fresh 1.7 mL Elution tube provided with the kit.

  2. Add 50 μL of Elution Buffer B to the column.

  3. 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.

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.

  1. 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.

  2. Thaw the desired barcodes at room temperature.

  3. 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.

  4. Thaw the LongAmp Hot Start Taq 2X Master Mix, spin down briefly, mix well by pipetting and place on ice.

  5. 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.

  6. 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

  7. Ensure the components are thoroughly mixed by pipetting and spin down briefly.

  8. 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.

  9. Mix the 16S barcodes by pipetting up and down 10 times. Transfer 10 μl of each 16S Barcode into respective sample-containing tubes.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. Incubate for 5 minutes at room temperature.

  15. Check your PCR product

    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.

  16. Pool all barcoded samples in equimolar ratios in a 1.5 ml tube.

    Optional

    Eppendorf DNA LoBind tubes are recommended.

  17. Resuspend the AMPure XP Beads (AXP) by vortexing.

  18. 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

  19. Incubate for 5 minutes at room temperature; gently mix by inversion every minute or so; or use a Hula mixer.

  20. Prepare 2 ml of fresh 80% ethanol in nuclease-free water.

  21. 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.

  22. 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.

  23. Repeat the previous step.

  24. 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.

  25. 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.

  26. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.

  27. 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.

  28. 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

  29. Add 0.5µl of diluted Rapid Adapter (RA) to the barcoded DNA.

  30. Mix gently by flicking the tube, and spin down.

  31. 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.

Nanopore

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

  1. 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.

  2. 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.

  3. Place the Flongle adapter into the MinION.

  4. Place the flow cell into the Flongle adapter, and press the flow cell down until you hear a click.

  5. Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Navigate to the start page and click Start sequencing.

  11. Fill in your experiment details, such as name and flow cell position and sample ID.

  12. Select the sequencing kit (16S Barcoding Kit 24 V14 (SQK-16S114.24)) used in the library preparation on the Kit page.

  13. Configure the sequencing parameters for your sequencing run or keep to the default settings on the Run options and Analysis tabs.

    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.

  14. On the Output page, set up the output parameters or keep to the default settings.

  15. 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.

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

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

    Bioinformatics

    In this example, we use the Continue as guest option.

  2. 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.

  3. Click on the Launch button to proceed with the workflow.

  4. 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.

  5. 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:

  1. 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.

  2. 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.

  3. 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).

  4. 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.

  5. 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.

  6. 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

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