Rapid sequencing DNA - 16S Barcoding Kit 24 V14 (SQK-16S114.24)

Version for device: Flongle

Other available device versions: MinION

1. Introduction to the 16S Barcoding Kit 24 V14

   This protocol describes how to carry out rapid barcoding of 16S amplicons using the 16S Barcoding Kit 24 V14 (SQK-16S114.24). Due to the presence of both highly conserved (adequate for universal primers and phylogenetic signal) and highly variant regions (different across species), the 16S rRNA gene is often used for sequence-based bacterial identification.

   The 16S Barcoding Kit 24 V14 enables access to rapid 16S sequencing for organism identification. By narrowing down to a specific region of interest, you can see all the organisms in the sample without sequencing unnecessary regions of the genome, making the test quicker and more economical. There are 24 unique barcodes, allowing you to pool up to 24 different samples in one sequencing experiment.

   After sequencing, you can perform downstream analysis using the EPI2ME 16S workflow (wf-16s) to classify 16S amplicons from your samples.

   Steps in the sequencing workflow:

   Prepare for your experiment

   You will need to:

   • Extract your gDNA, and check its length, quantity and purity using the Input DNA/RNA QC protocol. The quality checks performed during the protocol are essential in ensuring experimental success
   • Ensure you have your sequencing kit, the correct equipment and third-party reagents
   • Download the software for acquiring and analysing your data
   • Check your flow cell to ensure it has enough pores for a good sequencing run

   
   

   Library preparation

   The table below is an overview of the steps required in the library preparation, including timings and stopping points.

   Library preparation step	Process	Time	Stop option
   16S barcoded PCR amplification	Amplify the 16S gene using barcodes supplied in the kit	10 minutes + PCR	4°C overnight
   Barcoded sample pooling and bead clean-up	Quantify and pool the barcoded samples and perform a library clean-up using beads	15 minutes	4°C short-term storage or for repeated use, such as re-loading your flow cell. -80°C for single-use long-term storage.
   Adapter ligation	Attach the rapid sequencing adapters to the to the DNA ends.	5 minutes	We strongly recommend sequencing your library as soon as it is adapted.
   Priming and loading the flow cell	Prime the flow cell and load the prepared DNA library for sequencing	5 minutes

   

   Sequencing and analysis

   You will need to:

   • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
   • Optional: Start the EPI2ME software and select the wf-16S workflow
2. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • 16S Barcoding Kit 24 V14 (SQK-16S114.24)
   • Flongle Sequencing Expansion (EXP-FSE002)
   • R10.4.1 flow cells (FLO-FLG114)
   • Rapid Adapter Auxiliary V14 (EXP-RAA114)
   • Sequencing Auxiliary Vials V14 (EXP-AUX003)
   • MinION Mk1C - MinION Mk1C IT requirements document
   • MinION Mk1B - MinION IT Requirements document
   • GridION - GridION IT requirements document

1. Important Flongle Sequencing Expansion (EXP-FSE002)

   There are three buffers that come into direct contact with a flow cell at point of loading (SB: Sequencing Buffer, FCF: Flow Cell Flush and LIB: Library Beads or LIS: Library Solution). When looking at these buffers, we found that there are a very low level of contaminants seeping out of the plastic vials that impacts the robustness of the Flongle flow cell system (MinION and PromethION are not impacted by this).

   We have found that when storing these buffers in glass vials instead of plastic, incidence of deterioration is reduced.

   

   To rapidly deploy this to Flongle users, we have produced a Flongle Sequencing Expansion (EXP-FSE002) with these three components in glass vials, which can perform 12 Flongle flow cell loads in total.

   To load a library onto your Flongle flow cell, you will need to use the following components:

   Flongle Sequencing Expansion (EXP-FSE002) components
   - Sequencing Buffer (SB)
   - Flow Cell Flush (FCF)
   - Library Beads (LIB) or Library Solution (LIS)

   Sequencing Kit components
   - Flow Cell Tether (FCT)

   Oxford Nanopore Technologies deem the useful life of the Flow Cell Expansion to be 6 months from receipt by the customer.

2. For this protocol, you will need 10 ng high molecular weight genomic DNA per barcode.

   For optimal output, we currently do not recommend using fewer than 4 barcodes. If you wish to multiplex less than 4 samples, please ensure you split your sample(s) across multiple barcodes so at least 4 barcodes are run (e.g. for 2 samples, use 16S Barcode Primers 01-02 for sample A, and 16S Barcode Primers 03-04 for sample B). Please note that the required sample input for each barcode is 10 ng gDNA.

3. Third-party reagents

   We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

   For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

4. Check your flow cell

   We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

   Flow cell	Minimum number of active pores covered by warranty
   Flongle Flow Cell	50
   MinION/GridION Flow Cell	800
   PromethION Flow Cell	5000

5. 16S Barcoding Kit 24 V14 contents

   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
   16S Barcode Primers 01-24	1-24	-	2 plates, 3 sets of barcodes per plate	15 μl per well
   Rapid Adapter	RA	Green	1	15
   Adapter Buffer	ADB	Clear	1	100
   AMPure XP Beads	AXP	Clear cap, light teal label	1	6,000
   Elution buffer	EB	Black	1	1,500
   EDTA	EDTA	Blue	1	700
   Sequencing Buffer	SB	Red	1	700
   Library Beads	LIB	Pink	1	600
   Library Solution	LIS	White cap, pink label	1	600
   Flow Cell Flush	FCF	Clear cap, light blue label	1	8,000
   Flow Cell Tether	FCT	Purple	1	200

   Note: This product contains AMPure XP Reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

6. Flongle Sequencing Expansion (EXP-FSE002) contents

   

   Name	Acronym	Cap colour	Number of vials	Fill volume per vial (µl)
   Sequencing Buffer	SB	Blue	1	250
   Library Beads	LIB	Blue	1	200
   Library Solution	LIS	Blue	1	200
   Flow Cell Flush	FCF	Blue	1	1,600

   Oxford Nanopore Technologies deem the useful life of the Flow Cell Expansion to be 6 months from receipt by the customer.

1. Minimum 16S Barcode Primers use requirements

   For optimal output, we currently do not recommend using fewer than 4 barcodes. If you wish to multiplex less than 4 samples, please ensure you split your sample(s) across barcodes so a minimum of 4 barcodes are run:

   • For 1 sample, run your sample across 4 barcodes (e.g. 16S Barcode Primers 01-04 using 10 ng of sample A per barcode)
   • For 2 samples, run each sample across two barcodes. (e.g. 16S Barcode Primers 01-02 for sample A, and 16S Barcode Primers 03-04 for sample B)
   • For 3 samples, run two samples individually and one across 2 barcodes. (e.g. 16S Barcode Primer 01 and 16S Barcode Primer 02 for sample A and B respectively, and 16S Barcode Primers 03-04 for sample C)

   Please note that the required sample input for each barcode is 10 ng gDNA.

2. Check your flow cell.

   We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.

   See the flow cell check instructions in the MinKNOW protocol for more information.

3. 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.

   16S barcode plate layout:

   

4. Important The 96-well plates are designed to break in one direction only. Strips, or multiple strips, of eight wells/barcodes can be removed from the plate at any one time.
5. Thaw the desired barcodes at room temperature.
6. Briefly centrifuge barcodes in a microfuge to make sure the liquid is at the bottom of the tubes and place on ice.
7. Thaw the LongAmp Hot Start Taq 2X Master Mix, spin down briefly, mix well by pipetting and place on ice.
8. Prepare the DNA in nuclease-free water.
   • Transfer 10 ng of each genomic DNA sample into a 0.2 ml thin-walled PCR tube
   • Adjust the volume to 15 μl with nuclease-free water
   • Mix thoroughly by flicking avoiding unwanted shearing
   • Spin down briefly in a microfuge

9. 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)	15 μl
   LongAmp Hot Start Taq 2X Master Mix	25 μl
   Total	40 μl

   Note: If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.

10. Ensure the components are thoroughly mixed by pipetting and spin down briefly.
11. 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.
12. Using a multichannel pipette, mix the 16S barcodes by pipetting up and down 10 times. Transfer 10 μl of each 16S Barcode into respective sample-containing tubes.
13. Ensure the components are thoroughly mixed by pipetting the contents of the tubes 10 times and spin down.

    Note: Mix gently to minimise introducing air bubbles to the reactions.

14. Amplify using the following cycling conditions:

    Cycle step	Temperature	Time	No. of cycles
    Initial denaturation	95 °C	1 min	1
    Denaturation	95 °C	20 secs	25
    Annealing	55 °C	30 secs	25
    Extension	65 °C	2 mins	25
    Final extension	65 °C	5 mins	1
    Hold	4 °C	∞

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

    Reagent	1. Thaw at room temperature	2. Briefly spin down	3. 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

    Note: Once thawed, keep all reagents on ice.

16. Add 4 µl of EDTA to each barcoded sample, mix thorougly by pipetting and spin down briefly.
17. Tip EDTA is added at this step to stop the reaction.
18. Incubate for 5 minutes at room temperature.
19. Quantify 1 µl of each barcoded sample using a Qubit fluorometer (or equivalent) for QC check.
20. Pool all barcoded samples in equimolar ratios in a 1.5 ml Eppendorf DNA LoBind tube.

    Note: Please ensure you have quantified your samples prior to this step and take forward an equimolar concentration of each of the samples for optimal barcode balancing.
    Samples may vary in concentration following the barcoded PCR, therefore the volume of each barcoded sample added to the pool will be different.

21. Resuspend the AMPure XP Beads (AXP) by vortexing.
22. 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	37.5 μl	75 μl	150 μl	300 μl	600 μl
    Volume of AMPure XP Beads (AXP)	22.5 μl	45 μl	90 μl	180 μl	360 μl

    Note: Table contains example volumes for reference. Please adjust the volume of AMPure XP Beads (AXP) added for the volume of your barcoded sample pool to ensure a 0.6X volume ratio.

23. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
24. Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
25. Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, and pipette off the supernatant.
26. 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.
27. Repeat the previous step.
28. 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.
29. 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.
30. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
31. Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
    • Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
    • Dispose of the pelleted beads

32. Quantify 1 µl of eluted sample using a Qubit fluorometer.
33. Transfer 50 fmol of your eluted sample into a clean 1.5 ml Eppendorf DNA LoBind tube. Make up the volume to 5.5 µl with Elution Buffer (EB).
34. In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:

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

35. Add 0.5 µl of diluted Rapid Adapter (RA) to the barcoded DNA.
36. Mix gently by flicking the tube, and spin down.
37. Incubate the reaction for 5 minutes at room temperature.
38. End of Step The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

1. Important Please note, this kit is only compatible with R10.4.1 flow cells (FLO-FLG114).
2. Important Flongle Sequencing Expansion (EXP-FSE002)

   There are three buffers that come into direct contact with a flow cell at point of loading (SB: Sequencing Buffer, FCF: Flow Cell Flush and LIB: Library Beads or LIS: Library Solution). When looking at these buffers, we found that there are a very low level of contaminants seeping out of the plastic vials that impacts the robustness of the Flongle flow cell system (MinION and PromethION are not impacted by this).

   We have found that when storing these buffers in glass vials instead of plastic, incidence of deterioration is reduced.

   

   To rapidly deploy this to Flongle users, we have produced a Flongle Sequencing Expansion (EXP-FSE002) with these three components in glass vials, which can perform 12 Flongle flow cell loads in total.

   To load a library onto your Flongle flow cell, you will need to use the following components:

   Flongle Sequencing Expansion (EXP-FSE002) components
   - Sequencing Buffer (SB)
   - Flow Cell Flush (FCF)
   - Library Beads (LIB) or Library Solution (LIS)

   Sequencing Kit components
   - Flow Cell Tether (FCT)

   Oxford Nanopore Technologies deem the useful life of the Flow Cell Expansion to be 6 months from receipt by the customer.

3. Important Do NOT touch the reverse side of the Flongle flow cell array or the contact pads on the Flongle adapter. ALWAYS wear gloves when handling Flongle flow cells and adapters to avoid damage to the flow cell or adapter.

   

4. The diagram below shows the components of the Flongle flow cell:

   

   The seal tab, air vent, waste channel, drain port and sample port are visible here. The sample port, drain port and air vent only become accessible once the seal tab is peeled back.

5. 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.
6. In a fresh 1.5 ml Eppendorf DNA LoBind tube, mix 117 µl of Flow Cell Flush (FCF) with 3 µl of Flow Cell Tether (FCT) and mix by pipetting.
7. Place the Flongle adapter into the MinION or one of the five GridION positions.

   The adapter should sit evenly and flat on the MinION Mk1B or GridION platform. This ensures the flow cell assembly is flat during the next stage.

8. Important The adapter needs to be plugged into your device, and the device should be plugged in and powered on before inserting the Flongle flow cell.

   

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

   The flow cell should sit evenly and flat inside the adapter, to avoid any bubbles forming inside the fluidic compartments.

   

10. Important How to prime and load a Flongle flow cell

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

    1. Lift up the seal tab:
       
       
       

    2. Pull the seal tab to open access to the sample port:
       
       
       

    3. Hold the seal tab open by using adhesive on the tab to stick to the MinION Mk 1B lid:
       

12. 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.

    

13. 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.

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

14. 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

15. 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.

    

16. Seal the Flongle flow cell using the adhesive on the seal tab, as follows:

    1. Stick the transparent adhesive tape to the sample port.
       
       
       

    2. Replace the top (Wheel icon section) of the seal tab to its original position.
       

17. End of Step Close the device lid and set up a sequencing run on MinKNOW.

1. How to start sequencing

   Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

   MinKNOW can be used and set up to sequence in multiple ways:

   • On a computer either direcly or remotely connected to a sequencing device.
   • Directly on a GridION, MinION Mk1C or PromethION 24/48 sequencing device.

   For more information on using MinKNOW on a sequencing device, please see the device user manuals:

   • MinION Mk1C user manual
   • MinION Mk1B user manual
   • GridION user manual

   ---

   To start a sequencing run on MinKNOW:

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

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

   3. Select the sequencing kit used in the library preparation on the Kit page.
   

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

   Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.

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

   6. Click Start on the Review page to start the sequencing run.
   

2. Data analysis after sequencing

   After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

   In the Downstream analysis section, we outline further options for analysing your data.

1. Post-basecalling analysis

   We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a desktop application. EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis.

   Further information about the available EPI2ME workflows are available here, along with the Quick Start Guide to start your first bioinformatic workflow.

   The 16S workflow (wf-16s) is a Nextflow workflow leveraging the power of wf-metagenomics for identification of the origin of reads from targeted amplicon sequencing. The workflow has two modes of operation, it can use either kraken2 or minimap2 to determine the origin of reads.

   More information on the EPI2ME 16S workflow (wf-16s) can be found here.

   For installation instructions please click here.

2. Additional options for further analysing your basecalled data include:

   1. Research analysis tools

   Oxford Nanopore Technologies' Research division has created a number of analysis tools, that are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

   2. Community-developed analysis tools

   If a data analysis method for your research question is not provided in any of the resources above, please refer to the Bioinformatics section of the Resource centre. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

1. Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

   We also have an FAQ section available on the Nanopore Community Support section.

   If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

2. Low sample quality

   Observation	Possible cause	Comments and actions
   Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2)	The DNA extraction method does not provide the required purity	The effects of contaminants are shown in the Contaminants document. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step.
   Low RNA integrity (RNA integrity number <9.5 RIN, or the rRNA band is shown as a smear on the gel)	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page.
   RNA has a shorter than expected fragment length	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page. We recommend working in an RNase-free environment, and to keep your lab equipment RNase-free when working with RNA.

3. Low DNA recovery after AMPure bead clean-up

   Observation	Possible cause	Comments and actions
   Low recovery	DNA loss due to a lower than intended AMPure beads-to-sample ratio	1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample. 2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size will be lost during the clean-up.
   Low recovery	DNA fragments are shorter than expected	The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use.
   Low recovery after end-prep	The wash step used ethanol <80%	DNA will be eluted from the beads when using ethanol <80%. Make sure to use the correct percentage.

1. Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

   We also have an FAQ section available on the Nanopore Community Support section.

   If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

2. Fewer pores at the start of sequencing than after Flow Cell Check

   Observation	Possible cause	Comments and actions
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	An air bubble was introduced into the nanopore array	After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video.
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	The flow cell is not correctly inserted into the device	Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	Contaminations in the library damaged or blocked the pores	The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

3. MinKNOW script failed

   Observation	Possible cause	Comments and actions
   MinKNOW shows "Script failed"		Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

4. Pore occupancy below 40%

   Observation	Possible cause	Comments and actions
   Pore occupancy <40%	Not enough library was loaded on the flow cell	10–50 fmol of good quality library can be loaded on to a MinION Mk1B/GridION flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
   Pore occupancy close to 0	The Rapid PCR Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA	Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents.
   Pore occupancy close to 0	No tether on the flow cell	Tethers are added during flow cell priming (FCT tube). Make sure FCT was added to FCF before priming.

5. Shorter than expected read length

   Observation	Possible cause	Comments and actions
   Shorter than expected read length	Unwanted fragmentation of DNA sample	Read length reflects input DNA fragment length. Input DNA can be fragmented during extraction and library prep. 1. Please review the Extraction Methods in the Nanopore Community for best practice for extraction. 2. Visualise the input DNA fragment length distribution on an agarose gel before proceeding to the library prep. In the image above, Sample 1 is of high molecular weight, whereas Sample 2 has been fragmented. 3. During library prep, avoid pipetting and vortexing when mixing reagents. Flicking or inverting the tube is sufficient.

6. Large proportion of unavailable pores

   Observation	Possible cause	Comments and actions
   Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot) The pore activity plot above shows an increasing proportion of "unavailable" pores over time.	Contaminants are present in the sample	Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores stays large or increases: 1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be performed, or 2. Run several cycles of PCR to try and dilute any contaminants that may be causing problems.

7. Large proportion of inactive pores

   Observation	Possible cause	Comments and actions
   Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged)	Air bubbles have been introduced into the flow cell	Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice
   Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides, typically associate with plant genomic DNA. 1. Please refer to the Plant leaf DNA extraction method. 2. Clean-up using the QIAGEN PowerClean Pro kit. 3. Perform a whole genome amplification with the original gDNA sample using the QIAGEN REPLI-g kit.
   Large proportion of inactive/unavailable pores	Contaminants are present in the sample	The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

8. Temperature fluctuation

   Observation	Possible cause	Comments and actions
   Temperature fluctuation	The flow cell has lost contact with the device	Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services.

9. Failed to reach target temperature

   Observation	Possible cause	Comments and actions
   MinKNOW shows "Failed to reach target temperature"	The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating)	MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW. Please refer to this FAQ for more information on MinION temperature control.