cDNA-PCR Sequencing (SQK-PCS111)

Version for device: MinION

1. Important This is a Legacy product

   This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com. For further information on please see the product update page.

2. cDNA-PCR Sequencing Kit features

   This kit is highly recommended for users who:

   • Would like to identify and quantify full-length transcripts
   • Are looking for a faster and simpler method for cDNA synthesis: ~210 minutes library prep + variable time for PCR
   • Want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
   • Wish to start from total RNA or have a low starting amount of RNA
   • Would like to generate high amounts of cDNA data
3. Introduction to cDNA-PCR protocol

   This protocol describes how to carry out sequencing of cDNA using a strand-switching method and the cDNA-PCR Sequencing Kit (SQK-PCS111). During the strand-switching step, a UMI is incorporated, before the double-stranded cDNA is amplified by PCR using primers containing 5' tags. The Rapid Sequencing Adapters are then added to the amplified sample.

   A control experiment can be completed first using RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as your input to troubleshoot your library preparation or to become familiar with the protocol.

   Steps in the sequencing workflow:

   Prepare for your experiment

   You will need to:

   • Extract your RNA, and check its length, quantity and purity. 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

   You will need to:

   • Using the strand-switching protocol, prepare full-length cDNAs from Poly(A)+ RNA (or total RNA)
   • Amplify the cDNAs by PCR, adding rapid attachment primers during the PCR step
   • Attach sequencing adapters to the PCR products
   • Prime the flow cell, and load your cDNA library into the flow cell

   

   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 a workflow for further analysis, e.g. metagenomic analysis or drug resistance mapping
4. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • cDNA-PCR Sequencing Kit (SQK-PCS111)
   • R9.4.1 flow cells (FLO-MIN106)
   • Flow Cell Wash Kit (EXP-WSH004)
   • RNA Control Expansion (EXP-RCS001)

1. For this protocol, you will need 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA.
2. Input RNA

   It is important that the input RNA meets the quantity and quality requirements. Using too little or too much RNA, or RNA of poor quality (e.g. fragmented or containing chemical contaminants) can affect your library preparation.

   For instructions on how to perform quality control of your RNA sample, please read the Input DNA/RNA QC protocol.

   For further information on using RNA as input, please read the links below.

   • Polyadenylation of non-poly(A) transcripts using E. coli poly(A) polymerase
   • RNA contaminants
   • RNA stability
   • RNA Integrity Number (RIN)
   • Enrichment of polyadenylated RNA molecules

   These documents can also be found in the DNA/RNA Handling page.

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. Important Rapid Adapter T (RAP T) used in this kit and protocol is not interchangeable with other sequencing adapters.
5. cDNA-PCR Sequencing Kit (SQK-PCS111) contents

   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
   Strand Switching Primer II	SSPII	Violet	1	20 µl
   RT Primer	RTP	Yellow	1	10 µl
   cDNA RT Adapter	CRTA	Amber	1	10 µl
   Rapid Adapter T	RAP T	Green	1	10 µl
   Annealing Buffer	AB	Orange	1	10 µl
   cDNA Primer	cPRM	White cap, grey label	1	40 µl
   Elution Buffer	EB	Black	1	500 µl
   Short Fragment Buffer	SFB	Clear	1	1,800 µl
   Sequencing Buffer II	SBII	Red	1	500 µl
   Loading Beads II	LBII	Pink	1	360 µl
   Loading Solution	LS	White cap, pink label	1	400 µl
   Flush Buffer	FB	Blue	6	1,170 µl
   Flush Tether	FLT	White cap, purple label	1	200 µl

6. RNA Control Expansion (EXP-RCS001)

   The RNA Control Expansion (EXP-RCS001) provides users with RNA Control Sample (RCS) to replace their sample input when performing a control experiment for troubleshooting purposes in the cDNA-PCR Sequencing (SQK-PCS111) and the PCR-cDNA Barcoding Kit (SQK-PCB111.24).

   

   Name	Acronym	Number of vials	Cap colour	Fill volume per vial (µl)
   RNA Control Sample	RCS	3	Yellow	25

1. MinION Mk1C IT requirements

   The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse nanopore data. Read more in the MinION Mk1C IT requirements document.

2. Software for nanopore sequencing

   MinKNOW

   The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

   For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

   EPI2ME (optional)

   The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

   For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.

3. 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

1. Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.

   Reagent	1. Thaw at room temperature	2. Briefly spin down	3. Mix well by pipetting
   cDNA RT Adapter (CRTA)	✓	✓	✓
   Annealing Buffer (AB)	✓	✓	✓
   Short Fragment Buffer (SFB)	✓	✓	✓
   RT Primer (RTP)	✓	✓	✓
   Strand Switching Primer II (SSPII)	✓	✓	✓
   NEBNext® Quick Ligation Reaction Buffer	✓	✓	Mix by vortexing
   T4 DNA Ligase 2M U/ml	Not frozen	✓	✓
   RNaseOUT	Not frozen	✓	✓
   Lambda Exonuclease	Not frozen	✓	✓
   Uracil-Specific Excision Reagent (USER)	Not frozen	✓	✓
   10 mM dNTP solution	✓	✓	✓
   Maxima H Minus Reverse Transcriptase	Not frozen	✓	✓
   Maxima H Minus 5x RT Buffer	✓	✓	Mix by vortexing

2. Important It is important that the NEBNext Quick Ligation Reaction Buffer is mixed well by vortexing.

   Check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise all precipitate.

3. Optional Action

   To run a control experiment, replace your sample input with 10 μl diluted RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as follows:

   • Thaw the RNA Control Sample (RCS) at room temperature, briefly spin down and mix well by pipetting.
   • Dilute the RNA Control Sample (RCS) in a 1.5 ml Eppendorf DNA LoBind tube as follows:

   Reagent	Volume
   RNA Control Sample (RCS)	1 μl
   Nuclease-free water	14 μl
   Total	15 μl

   Note: This will provide enough volume for 3 samples, adjust your volumes accordingly for the number of samples you wish to run in your control experiment.

   • Mix thoroughly by pipetting 10-20 times and briefly spin down.
   • Take forward 4 μl of the diluted RNA Control Sample (RCS) per sample and make up each volume to 10 μl with nuclease-free water as follows:

   Reagent	Volume
   Diluted RNA Control Sample (RCS)	4 μl
   Nuclease-free water	6 μl
   Total volume per sample	10 μl

   • Mix by flicking the tube and spin down.
   • Use the 10 μl of diluted RNA Control Sample (RCS) as your RNA input.
4. Prepare the RNA in nuclease-free water.
   • Transfer 4 ng Poly(A)+ RNA, or 200 ng total RNA into a 1.5 ml Eppendorf DNA LoBind tube
   • Adjust the volume to up to 10 μl with nuclease-free water
   • Mix by flicking the tube to avoid unwanted shearing
   • Spin down briefly in a microfuge

5. Prepare the following reaction in a 0.2 ml PCR tube:

   Reagent	Volume
   RNA	10 μl
   cDNA RT Adapter (CRTA)	1 μl
   Annealing Buffer (AB)	1 μl
   Total volume	12 μl

6. Tip The cDNA RT Adapter (CRTA) is a double stranded adapter with a poly(T) overhang which anneals to the very end of the poly(A) tail of the RNA strand. This ensures that the full length of the RNA is reverse transcribed and that the poly(A) length can be estimated accurately. Annealing Buffer (AB) has been included to improve CRTA ligation.
7. Ensure the components are thoroughly mixed by flicking the tube and spin down.
8. Incubate the reaction in the thermal cycler at 60°C for 5 minutes, then cool for 10 minutes at room temperature.
9. To the same 0.2 ml PCR tube, add the following:

   Reagent	Volume
   RNA sample (from previous step)	12 μl
   NEBNext® Quick Ligation Reaction Buffer	3.6 μl
   T4 DNA Ligase 2M U/ml	1.4 μl
   RNaseOUT	1 μl
   Total volume (including all reagents)	18 μl

10. Ensure the components are thoroughly mixed by flicking the tube and spin down.
11. Incubate for 10 minutes at room temperature.
12. To the same 0.2 ml PCR tube, add the following:

    Reagent	Volume
    Lambda Exonuclease	1 μl
    Uracil-Specific Excision Reagent (USER)	1 μl
    Total volume (including all reagents)	20 μl

13. Tip The Lambda Exonuclease and Uracil-Specific Excision Reagent (USER) are third-party reagents used in the preparation of the reverse transcription step. Lambda Exonuclease and USER digest the bottom strand of the ligated CRTA so that the RT Primer (RTP) can bind the CRTA sequence as a primer for the reverse transcription of the RNA.
14. Ensure the components are thoroughly mixed by flicking the tube and spin down.
15. Incubate for 15 minutes at 37°C in the thermal cycler.
16. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
17. Resuspend the RNase-free XP beads by vortexing.
18. Add 36 µl of resuspended RNase-free XP beads to the reaction and mix gently by flicking the tube.
19. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
20. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
21. Keep the tube on the magnet and wash the beads with 100 µl of Short Fragment Buffer (SFB) without disturbing the pellet. Remove the SFB using a pipette and discard.
22. Repeat the previous step.
23. Spin down and place the tube back on the magnet. Pipette off any residual buffer. Briefly allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
24. Remove the tube from the magnetic rack and resuspend pellet in 12 µl of nuclease-free water.
25. Incubate at room temperature for 10 minutes.
26. Pellet the beads on a magnet until the eluate is clear and colourless.
27. Remove and retain 12 µl of eluate into a clean 0.2 ml thin-walled PCR tube.
28. To the same 0.2 ml PCR tube, add the following:

    Reagent	Volume
    Eluted sample (from previous step)	12 μl
    RT Primer (RTP)	1 μl
    dNTPs (10 mM)	1 μl
    Total volume (including all reagents)	14 μl

29. Tip RT Primer (RTP) is a single stranded primer and binds upstream of the poly(A) tail of the RNA transcript to prime for reverse transcription.
30. Ensure the components are thoroughly mixed by flicking the tube and spin down.
31. Incubate the reaction for 15 minutes at room temperature.
32. To the same 0.2 ml PCR tube, add the following:

    Reagent	Volume
    RT primed RNA (from previous step)	14 μl
    Maxima H Minus 5x RT Buffer	4.5 μl
    RNaseOUT	1 μl
    Strand Switching Primer II (SSPII)	2 μl
    Total (including all reagents)	21.5 μl

33. Tip Strand Switching Primer II (SSPII) base pairs to the deoxycytidine present at the 5' end of the first cDNA strand synthesised. This allows the reverse transcriptase to "strand-switch" for synthesis of the second cDNA strand.
34. Ensure the components are thoroughly mixed by flicking the tube and spin down.
35. Incubate at 42°C for 2 minutes in the thermal cycler.
36. Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 22.5 µl.
37. Ensure the components are thoroughly mixed by flicking the tube and spin down.
38. Incubate using the following protocol using a thermal cycler:

    Cycle step	Temperature	Time	No. of cycles
    Reverse transcription and strand-switching	42°C	90 mins	1
    Heat inactivation	85°C	5 mins	1
    Hold	4°C	∞

39. End of Step Take your samples forward into the next step. However, at this point it is also possible to store the sample at -20°C overnight.

1. Important The 22.5 μl of reverse-transcribed sample is used to make 4x 50 μl PCR reactions which will be pooled at a later stage, with 5 μl of reverse-transcribed sample in each PCR reaction. Do NOT use all 22.5 μl of the reverse transcription reaction in a single PCR reaction.

   Reverse transcriptase is a PCR inhibitor and the RT material must be diluted enough for PCR to take place.

2. Thaw the cDNA Primer (cPRM), Elution Buffer (EB). LongAmp Hot Start Taq 2X Master Mix and Exonuclease I at room temperature, spin down and pipette mix. Store the reagents on ice.
3. Spin down the reverse-transcribed RNA sample.
4. Prepare four fresh 0.2 ml PCR tubes and add 5 μl of reverse-transcribed sample per tube.
5. In each of the 0.2 ml PCR tubes containing the reverse-transcribed sample, prepare the following reaction at room temperature:

   Reagent	Volume
   Reverse-transcribed sample (from previous step)	5 μl
   cDNA Primer (cPRM)	1.5 μl
   Nuclease-free water	18.5 μl
   2x LongAmp Hot Start Taq Master Mix	25 μl
   Total (including all reagents)	50 μl

6. Mix gently by pipetting.
7. Amplify using the following cycling conditions.

   Cycle step	Temperature	Time	No. of cycles
   Initial denaturation	95°C	30 secs	1
   Denaturation	95°C	15 secs	10-18*
   Annealing	62°C	15 secs	10-18*
   Extension	65°C	60 secs per kb	10-18*
   Final extension	65°C	6 mins	1
   Hold	4°C	∞

   *We recommend 14 cycles as a starting point. However, the number of cycles can be adjusted between the values shown according to experimental needs.

   For further information, please read The effect of varying the number of PCR cycles in the PCR-cDNA Sequencing Kit document.

8. Add 1 μl Exonuclease I directly to each PCR tube. Mix by pipetting.
9. Tip Exonuclease I is added to remove any excess primers which have not successfully annealed.
10. Incubate the reaction at 37°C for 15 minutes, followed by 80°C for 15 minutes in the thermal cycler.
11. Pool the four PCR reactions (total 204 μl) in a clean 1.5 ml Eppendorf DNA LoBind tube.
12. Resuspend the AMPure XP beads by vortexing.
13. Add 160 µl of resuspended AMPure XP beads to the reaction.
14. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
15. Prepare 1 ml of fresh 70% ethanol in nuclease-free water.
16. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
17. Keep the tube on the magnet and wash the beads with 500 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
18. Repeat the previous step.
19. 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.
20. Remove the tube from the magnetic rack and resuspend pellet in 12 µl of Elution Buffer (EB).
21. Incubate at room temperature for 10 minutes.
22. Pellet the beads on the magnet until the eluate is clear and colourless.
23. Remove and retain 12 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
    • Remove and retain the eluate which contains the cDNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
    • Dispose of the pelleted beads

24. For each sample, analyse 1 µl of the amplified cDNA for size, quantity and quality using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) for a QC check.
25. Important Sometimes a high-molecular weight product is visible in the wells of the gel when the PCR products are run, instead of the expected smear. These libraries are typically associated with poor sequencing performance. We have found that repeating the PCR with fewer cycles can remedy this.
26. Take forward 15-25 fmol of amplified cDNA and make the volume up to 11 μl in Elution Buffer (EB).

    Mass	Molarity if fragment length = 0.5 kb	Molarity if fragment length = 1.5 kb	Molarity if fragment length = 3 kb
    5 ng	16 fmol	5 fmol	3 fmol
    10 ng	32 fmol	11 fmol	5 fmol
    15 ng	49 fmol	16 fmol	8 fmol
    20 ng	65 fmol	22 fmol	11 fmol
    25 ng	81 fmol	27 fmol	13 fmol
    50 ng	154 fmol	51 fmol	26 fmol

    If the quantity of amplified cDNA is above 25 fmol, the remaining cDNA can be frozen and stored for another sequencing experiment (in this case, library preparation would start from the Adapter Addition step). We recommend avoiding multiple freeze-thaw cycles to prevent DNA degradation.

    The new sequencing adapter used in Kit 11 chemistry has a higher capture rate, enabling lower flow cell loading amounts to give optimal pore occupancy.

27. Tip Library storage recommendations

    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.

1. Important Rapid Adapter T (RAP T) used in this kit and protocol is not interchangeable with other sequencing adapters.
2. Spin down the Rapid Adapter T (RAP T) and place on ice.
3. Add 1 μl of Rapid Adapter T (RAP T) to the amplified cDNA library.
4. Mix well by pipetting and spin down.
5. Incubate the reaction for 5 minutes at room temperature.
6. Spin down briefly.
7. End of Step The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.
8. Tip Library storage recommendations

   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.

1. Tip Priming and loading a MinION flow cell

   We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.

2. Using the Loading Solution

   We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water.
   Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

3. Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down at room temperature.
4. To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
5. Open the MinION device lid and slide the flow cell under the clip.

   Press down firmly on the flow cell to ensure correct thermal and electrical contact.

   

   

6. Optional Action

   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.

7. Slide the flow cell priming port cover clockwise to open the priming port.

   

8. 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.
9. 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.

   

10. 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.

    

11. Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.
12. Important The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
13. In a new tube, prepare the library for loading as follows:

    Reagent	Volume per flow cell
    Sequencing Buffer II (SBII)	37.5 µl
    Loading Beads II (LBII) mixed immediately before use, or Loading Solution (LS), if using	25.5 µl
    DNA library	12 µl
    Total	75 µl

    Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII).

14. 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.

    

    

15. Mix the prepared library gently by pipetting up and down just prior to loading.
16. 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.

    

17. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.

    

    

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

    We recommend 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.

19. 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.

    

20. Caution The MinION Flow Cell Light Shield is not secured to the flow cell and careful handling is required after installation.
21. 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 directly 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 Mk1B user manual
   • MinION Mk1C 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 and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.

   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. Click Start to initiate the sequencing run.

2. Data analysis after sequencing

   After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

   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. After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.

   The Flow Cell Wash Kit protocol is available on the Nanopore Community.

2. Tip We recommend you to wash the flow cell as soon as possible after you stop the run. However, if this is not possible, leave the flow cell on the device and wash it the next day.
3. Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.

   Instructions for returning flow cells can be found here.

   Note: All flow cells must be flushed with deionised water before returning the product.

4. Important If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.

1. Post-basecalling analysis

   There are several options for further analysing your basecalled data:

   1. EPI2ME workflows

   For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

   2. Research analysis tools

   Oxford Nanopore Technologies' Research division has created a number of analysis tools, which 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.

   3. 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 resource centre and search for bioinformatics tools for your application. 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 <70%	DNA will be eluted from the beads when using ethanol <70%. 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	Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your 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 Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA	Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
   Pore occupancy close to 0	The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation	Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
   Pore occupancy close to 0	No tether on the flow cell	Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/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. Reduction in sequencing speed and q-score later into the run

   Observation	Possible cause	Comments and actions
   Reduction in sequencing speed and q-score later into the run	For Kit 9 chemistry (e.g. SQK-LSK109), fast fuel consumption is typically seen when the flow cell is overloaded with library (please see the appropriate protocol for your DNA library to see the recommendation).	Add more fuel to the flow cell by following the instructions in the MinKNOW protocol. In future experiments, load lower amounts of library to the flow cell.

9. 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.

10. 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.

11. Guppy – no input .fast5 was found or basecalled

    Observation	Possible cause	Comments and actions
    No input .fast5 was found or basecalled	input_path did not point to the .fast5 file location	The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
    No input .fast5 was found or basecalled	The .fast5 files were in a subfolder at the input_path location	To allow Guppy to look into subfolders, add the --recursive flag to the command

12. Guppy – no Pass or Fail folders were generated after basecalling

    Observation	Possible cause	Comments and actions
    No Pass or Fail folders were generated after basecalling	The --qscore_filtering flag was not included in the command	The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders.

13. Guppy – unusually slow processing on a GPU computer

    Observation	Possible cause	Comments and actions
    Unusually slow processing on a GPU computer	The --device flag wasn't included in the command	The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.