PCR tiling of African Swine Fever (ASF) virus (SQK-LSK109 with EXP-NBD104 or EXP-NBD114)

Version for device: MinION

1. Important This is a Legacy product

   This kit is available on the Legacy page of the store. We are in the process of gathering data to support the upgrade of this protocol to our latest chemistry. Further information regarding protocol upgrades will be provided on the Community as soon as they are available over the next few months. For further information on please see the product update page.

2. Important This protocol is a work in progress, and some details are expected to change over time. Please make sure you always use the most recent version of the protocol.

   This protocol is based on the No part gets left behind: Tiled nanopore sequencing of whole ASFV genomes stitched together using Lilo by Amanda Warr et al., 2021.

3. Native Barcoding Expansion 1-12 and 13-24 features

   These kits are recommended for users who:

   • wish to multiplex samples to reduce price per sample
   • want to optimise their sequencing experiment for throughput
   • require control over read length
   • are interested in utilising upstream processes such as size selection or whole genome amplification
4. Introduction to the ASFV Native Barcoding protocol

   This protocol describes an efficient, low-cost method to sequence ASFV at scale. The method uses tiled PCR amplification of the virus to obtain good coverage, while enabling sample multiplexing to reduce run costs.

   The kits used are the Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114), in conjunction with the Ligation Sequencing Kit (SQK-LSK109). There are 24 unique barcodes if using both expansion kits, allowing the user to pool up to 24 different samples in one sequencing experiment. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

   Steps in the sequencing workflow:

   Prepare for your experiment

   You will need to:

   • Extract your DNA, 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

   Prepare your library

   You will need to:

   • Amplify the viral genome using tiled primers, purify and pool the amplicons
   • Repair the DNA, and prepare the DNA ends for barcode attachment
   • Ligate Native barcodes supplied in the kit to the DNA ends
   • Ligate sequencing adapters supplied in the kit to the DNA ends
   • Prime the flow cell, and load your DNA library into the flow cell

   

   Sequencing

   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
   • Assemble and analyse the ASF genome using bioinformatics tools of your choice (recommendations included in the "Downstream analysis" sections of the protocol)
5. Important We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.
6. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • Ligation Sequencing Kit (SQK-LSK109)
   • Native Barcoding Expansions 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114)
   • FLO-MIN106 (R9.4.1) flow cells
   • Flow Cell Wash Kit (EXP-WSH004)

1. NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

   For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.

   Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.

2. Ligation Sequencing Kit (SQK-LSK109) contents

   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
   DNA CS	DCS	Yellow	1	50
   Adapter Mix	AMX	Green	1	40
   Ligation Buffer	LNB	Clear	1	200
   L Fragment Buffer	LFB	White cap, orange stripe on label	2	1,800
   S Fragment Buffer	SFB	Grey	2	1,800
   Sequencing Buffer	SQB	Red	2	300
   Elution Buffer	EB	Black	1	200
   Loading Beads	LB	Pink	1	360

3. Flow Cell Priming Kit (EXP-FLP002) contents

   

   Name	Acronym	Cap colour	No. of vial	Fill volume per vial (μl)
   Flush Buffer	FB	Blue	6	1,170
   Flush Tether	FLT	Purple	1	200

4. Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) contents

   EXP-NBD104 kit contents
   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
   Native Barcode 01-12	NB01-12	White	12	20
   Adapter Mix II	AMII	Green	1	40

   
   
   EXP-NBD114 kit contents
   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
   Native Barcode 13-24	NB13-24	White	12	20
   Adapter Mix II	AMII	Green	1	40

5. Native barcode sequences

   The native barcode sequences are the reverse complement of the corresponding barcode sequence in other kits. 24 unique barcodes are available in the Native Barcoding Expansion 1-12 and 13-24 (EXP-NBD104 and EXP-NBD114).

   Native Barcoding Expansion 1-12 and 13-24 (EXP-NBD104 and EXP-NBD114)

   Component	Forward sequence	Reverse sequence
   NB01	CACAAAGACACCGACAACTTTCTT	AAGAAAGTTGTCGGTGTCTTTGTG
   NB02	ACAGACGACTACAAACGGAATCGA	TCGATTCCGTTTGTAGTCGTCTGT
   NB03	CCTGGTAACTGGGACACAAGACTC	GAGTCTTGTGTCCCAGTTACCAGG
   NB04	TAGGGAAACACGATAGAATCCGAA	TTCGGATTCTATCGTGTTTCCCTA
   NB05	AAGGTTACACAAACCCTGGACAAG	CTTGTCCAGGGTTTGTGTAACCTT
   NB06	GACTACTTTCTGCCTTTGCGAGAA	TTCTCGCAAAGGCAGAAAGTAGTC
   NB07	AAGGATTCATTCCCACGGTAACAC	GTGTTACCGTGGGAATGAATCCTT
   NB08	ACGTAACTTGGTTTGTTCCCTGAA	TTCAGGGAACAAACCAAGTTACGT
   NB09	AACCAAGACTCGCTGTGCCTAGTT	AACTAGGCACAGCGAGTCTTGGTT
   NB10	GAGAGGACAAAGGTTTCAACGCTT	AAGCGTTGAAACCTTTGTCCTCTC
   NB11	TCCATTCCCTCCGATAGATGAAAC	GTTTCATCTATCGGAGGGAATGGA
   NB12	TCCGATTCTGCTTCTTTCTACCTG	CAGGTAGAAAGAAGCAGAATCGGA
   NB13	AGAACGACTTCCATACTCGTGTGA	TCACACGAGTATGGAAGTCGTTCT
   NB14	AACGAGTCTCTTGGGACCCATAGA	TCTATGGGTCCCAAGAGACTCGTT
   NB15	AGGTCTACCTCGCTAACACCACTG	CAGTGGTGTTAGCGAGGTAGACCT
   NB16	CGTCAACTGACAGTGGTTCGTACT	AGTACGAACCACTGTCAGTTGACG
   NB17	ACCCTCCAGGAAAGTACCTCTGAT	ATCAGAGGTACTTTCCTGGAGGGT
   NB18	CCAAACCCAACAACCTAGATAGGC	GCCTATCTAGGTTGTTGGGTTTGG
   NB19	GTTCCTCGTGCAGTGTCAAGAGAT	ATCTCTTGACACTGCACGAGGAAC
   NB20	TTGCGTCCTGTTACGAGAACTCAT	ATGAGTTCTCGTAACAGGACGCAA
   NB21	GAGCCTCTCATTGTCCGTTCTCTA	TAGAGAACGGACAATGAGAGGCTC
   NB22	ACCACTGCCATGTATCAAAGTACG	CGTACTTTGATACATGGCAGTGGT
   NB23	CTTACTACCCAGTGAACCTCCTCG	CGAGGAGGTTCACTGGGTAGTAAG
   NB24	GCATAGTTCTGCATGATGGGTTAG	CTAACCCATCATGCAGAACTATGC

6. ASFV primer sequences

   ASFV primer sequences described in the protocol are subject to change. Any updates can be found at ASFV Lilo GitHub page.

   Amplicon - primer pair	Forward sequence	Reverse sequence
   ASFV1	GGCGTTCATTTCACAAGATGC	ACGGCATCTAAGCAGCTCAATG
   ASFV2	CAGGCCGATATATCATTTCATCAATATTCA	ACCCAAAGCCCTGGAATCCTTA
   ASFV3	GCAAACCAAGTGACTCACCCTC	ATTGTATGACGTCGGGGCAGAT
   ASFV4	ACCTAGTAAAAGTCCTAGAAAAACCTTCA	CGCCATTGTTTTACACAGTCGC
   ASFV5	TCGAGATTTTATTATTTGGATGCATCATCA	GGACTGATGAAAGCCGTGAA
   ASFV6	TCCACGCGGTACTTGGCTCC	AGGCCTCGTTGGTGGAAAGGA
   ASFV7	AGGGCTGATGCAAATCTCTTTTTCA	TCTCCGATTTTCGCATGCCAAA
   ASFV8	AGTTTGCAAAGAGCCTAAAGATAGACT	AGCGTGGAACTGTAGATGACGA
   ASFV9	TGCAGAAACCGCAGATGAATGT	ATAGGATTAGATGCGACGCCCA
   ASFV10	GCATGTAGAGAGGTTTTGGTAGTCA	GGAAACAGCTGGAGAGTTGTGG
   ASFV11	TGGTTTTGAAATAAAATGCCTTCTACGG	GGAATGCATGGACGAAGAAGCA
   ASFV11 (alt)	CTATGGGATGGGAAGAGTGGTCAA	CGTCAACCGCCGCATTAGC
   ASFV12	TCCTTGGGAGTTACAGCGAAGA	AATGAAATCATTCGCGGCGAGT
   ASFV13	CAGACATTGGCAGTGATGGCTA	GAAATGCCGGGCCTTCTACAAA
   ASFV14	GCTACTCCCCCAAATATCACATATAATTGT	TTTTTCGTGTTGCTGTTCGGGA
   ASFV15	GGATGGCACCCTTCTCACAATC	TGCGTATGACCCGATGTTGTTG
   ASFV16	GTCGACTTCACAGGAACAACGG	ACCCGCTTTACACAAAACACGT
   ASFV17	TGGAATTTCCTGACGTGGCAAA	GCAACCGCTATTCCAAACAGGA
   ASFV18	AGTTGTTGTCCTAGACCGTGGCA	TGAAAAGGAGGGCACGATCC
   ASFV19	CCCGTATGCGGGCGTACTTT	TGGCCTCTTCTTTCCCCCGA
   ASFV20	GGCCGCAACATTTGTGTCAAAG	GCTCGCGAACAAATTACTCCCA
   ASFV21	GAATGGCAGCGATGATCTCAGG	TGCAGGGCAAGGGTATACTGAA
   ASFV22	TGGCGTCGTTTAACAGCTTGAT	GCTGGATGGCAAATCGGTTGTA
   ASFV23	AGGCGTGAAAATTCTTCTTCAAACA	AGACGTTTTAAGCTGCATGGCA
   ASFV24	GGCAGCAGGATCTTAAAACCGG	TGCATAATGCCCAGCTTTTCGT
   ASFV25	GCTGTTTAAGCGTTTCAAGCTGA	CTCCGCGGGGAACATTGTTTTA
   ASFV26	CCCTGGGAGGAGTCATCATGAA	GGTCATTGACTTTGGAAGCGCT
   ASFV27	ACTGTCTGCTAGACTCCCAGGA	CCCAAGAGGAGGAATGGTTTGC
   ASFV28	GCCCCCTAGCGTCACCGAAT	CCAAGCCTGCTGCGAAGCTC
   ASFV29	GACGCAATTTCGGCTGTTTTTAAAA	GACTTGGTCTCCGGCTCAAAAG
   ASFV30	GTTGGGGTGTTGGAGCGAATAA	TTCTGCTTACGGACGATGCAAC
   ASFV31	TAGTTGTGAAGCGTTCTCGGGT	GAGCACATGTTACTCGCCACTC
   ASFV32	GGACTTCTTATGCTCAGATGGGC	ACTGCTGCAGGCGTTAAACATT

1. MinION Mk1B IT requirements

   Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. Read more in the MinION Mk1B IT Requirements document.

2. 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.

3. 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.

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

1. Important Below is one example of a method to extract DNA from swine blood samples. However, users can instead use other options (e.g. commercially-available DNA extraction kits) if preferred. The authors have found that spin columns are not suitable for this protocol due to PCR inhibitors being carried over in the blood. This method is optimised for frozen blood samples; other methods may work with fresh blood.
2. The blood samples used will have been tested for ASFV by PCR prior to DNA extraction. Individual blood samples are preferred; however this protocol can also work with pooled blood samples from multiple animals.
3. Transfer 50 μl of whole blood to a 1.5 ml Eppendorf DNA LoBind tube.
4. Add 1 ml of lysis buffer to each sample.
5. Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
6. Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
7. Centrifuge at 7,500 rpm for 5 minutes. You should see a pellet form at the bottom of the tube.
8. Without disturbing the pellet, remove and discard the supernatant.
9. Add 700 μl of 5X TEN buffer to each sample.
10. Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
11. Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
12. Incubate the samples at 95°C for 5 minutes in a heat block or water bath.
13. Centrifuge at 7,500 rpm for 5 minutes. You should see a pellet form at the bottom of the tube.
14. Without disturbing the pellet, remove and discard the supernatant.
15. Resuspend each pellet in 700 μl 100% isopropanol.
16. Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
17. Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
18. Prepare sufficient fresh 70% ethanol in nuclease-free water.
19. Centrifuge the samples at 14,500 rpm for 15 minutes. You should see a white pellet form at the bottom of the tube.
20. Without disturbing the pellet, remove and discard the supernatant.
21. Wash the pellet by adding 500 μl of freshly-prepared 70% ethanol and pipetting up and down.
22. Centrifuge the samples at 11,500 rpm for 5 minutes.
23. Without disturbing the pellet, remove and discard the supernatant.
24. Allow the pellet to dry for ~30 seconds, ensuring there is no ethanol left in the sample. However, do not dry the pellet to the point of cracking.
25. Resuspend the pellet in 30 μl nuclease-free water. If it is difficult to resuspend in this volume, add more nuclease-free water.
26. End of Step The resuspended samples will be taken forward into the tiled DNA amplification and clean-up step. The extracted DNA can be used immediately or stored at 4°C for up to one week. For longer-term storage place at -20°C or -80°C.

1. Prepare the primers according to the manufacturer's instructions.
2. Pool the tiled primer pairs in Eppendorf or PCR tubes following these proportions. The final stock concentration should be 100 μM.

   Odd primer pool:

   Primer pair	Concentration
   3	0.75X
   5	2X
   7	1X
   9	1X
   11	1X
   11 alt	1X
   13	1X
   15	1X
   17	1.5X
   19	1X
   21	1X
   23	1X
   25	2X
   27	1X
   29	1X
   31	0.5X

   Even primer pool:

   Primer pair	Concentration
   2	1X
   4	2X
   6	0.5X
   8	1.5X
   10	2X
   12	2X
   14	2.5X
   16	1X
   18	1.5X
   20	1X
   22	1.5X
   24	1.5X
   26	1.5X
   28	0.75X
   30	1.5X
   32	1X

   Primer one:

   Primer number	Concentration
   1	1X

   Note: Due to the proximity to the telomeric sequence, primer 1 has a shorter amplicon length. The primer does not perform optimally in when pooled with others, therefore it is prepared separately.

3. Optional Action

   To conserve the DNA samples, dilute 1:10 using nuclease-free water. Unused sample should be stored at –20°C.
4. Immediately before setting up the PCR, dilute each primer pool 1:10 in nuclease-free water to make a 10 μM working stock.
5. For each sample, prepare one reaction corresponding to each primer pool in 0.2 ml PCR tubes:

   Note: For each sample you will have three reactions – odd primer pool, even primer pool, and primer 1 pool.

   Between each addition, pipette mix 10-20 times.

   Reagent	Volume
   Nuclease-free water	9 µl
   ASFV DNA (1:10 dilution)	2 µl
   Primer pool (1:10 dilution)	1.5 µl
   VeriFi HS Mix (PCRBIO)	12.5 µl
   Total	25 µl

   Note: we recommend to make a PCR master mix, either for individual primer pools for multiple samples or one for the three primers sets.

6. Mix well by pipetting and spin down.
7. Incubate in a thermocycler using the following program:

   Step	Temperature	Time	Cycles
   Initial denaturation	98°C	1 min	1
   Denaturation Annealing Extension	98°C 60°C 72°C	15 sec 15 sec 4 min 40 sec	40
   Final extension	72°C	5 min	1
   Hold	10°C	∞

8. Resuspend the AMPure XP beads by vortexing.
9. Add 10 µl of resuspended AMPure XP beads to each tube and mix by gently pipetting.
10. Incubate for 10 minutes at room temperature.
11. Prepare 50 ml of fresh 70% ethanol in nuclease-free water.
12. Spin down the tubes and pellet the beads on a magnet for 5 minutes. Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
13. Keep the tubes on the magnet and wash the beads in each well with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
14. Repeat the previous step.
15. Spin down and place the tubes 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.
16. Remove the tubes from the magnetic rack and resuspend each pellet in 15 µl nuclease-free water. Incubate for 2 minutes at room temperature.
17. Pellet the beads on a magnet until the eluate is clear and colourless.
18. Remove and retain 15 µl of eluate containing the DNA for each sample, in its three respective primer pools, into a clean tube.

    Note: At this point you should still have three tubes for each DNA sample.

19. Quantify 1 μl of each cleaned PCR product using a Qubit ds DNA BR assay.
20. Using the Qubit reads, pool each sample by quantity in the following proportions:

    Primer pool	Proportion
    Pool 1	48%
    Pool 2	50%
    Amplicon 1	2%

21. Tip If your pools consistently have a similar Qubit quantification across samples, in future experiments it is possible to omit the Qubit step and pool the samples by volume based on previous results.
22. End of Step Take forward the DNA sample pools into the DNA repair and end-prep step. However, at this point it is also possible to store the samples at 4°C overnight.

1. Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.

   For optimal performance, NEB recommend the following:

   1. Thaw all reagents on ice.

   2. Flick and/or invert the reagent tubes to ensure they are well mixed.
      
      Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.

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

   4. The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
      
      Note: It is important the buffers are mixed well by vortexing.

   5. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

2. Prepare the DNA in nuclease-free water.
   • Transfer 700 ng of amplicon DNA into a 1.5 ml Eppendorf DNA LoBind tube
   • Adjust the volume to 48 μl with nuclease-free water
   • Mix thoroughly by flicking the tube
   • Spin down briefly in a microfuge

3. Tip If there is not enough PCR product for the 700 ng pool, the end-prep reaction below can be carried out at half the volume.
4. In a 0.2 ml thin-walled PCR tube, mix the following:

   Between each addition, pipette mix 10-20 times

   Reagent	Volume
   DNA	48 µl
   NEBNext FFPE DNA Repair Buffer	3.5 µl
   Ultra II End-prep reaction buffer	3.5 µl
   Ultra II End-prep enzyme mix	3 µl
   NEBNext FFPE DNA Repair Mix	2 µl
   Total	60 µl

5. Mix well by pipetting and spin down.
6. Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
7. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
8. Resuspend the AMPure XP beads by vortexing.
9. Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube.
10. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
11. Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
12. Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
13. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
14. Repeat the previous step.
15. 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.
16. Remove the tube from the magnetic rack and resuspend the pellet in 25 µl nuclease-free water. Spin down and incubate for 5 minutes at room temperature.
17. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
18. Remove and retain 25 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
19. Optional Action

    Quantify 1 µl of eluted sample using a Qubit fluorometer.
20. End of Step Take forward the repaired and end-prepped DNA into the native barcode ligation step. However, at this point it is also possible to store the sample at 4°C overnight.

1. Select a unique barcode for every sample to be run together on the same flow cell, from the provided 24 barcodes. Up to 24 samples can be barcoded and combined in one experiment.
2. Thaw the native barcodes at room temperature. Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.
3. Dilute 500 ng of each end-prepped sample to be barcoded to 22.5 µl in nuclease-free water.
4. Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:

   Reagent	Volume
   500 ng end-prepped DNA	22.5 µl
   Native Barcode	2.5 µl
   Blunt/TA Ligase Master Mix	25 µl
   Total	50 µl

5. Mix by pipetting up and down 10-20 times and spin down.
6. Incubate the reaction for 10 minutes at room temperature.
7. Resuspend the AMPure XP beads by vortexing.
8. Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
9. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
10. Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
11. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
12. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
13. Repeat the previous step.
14. 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.
15. Remove the tube from the magnetic rack and resuspend the pellet in 26 µl nuclease-free water. Incubate for 2 minutes at room temperature.
16. Pellet the beads on a magnet until the eluate is clear and colourless.
17. Remove and retain 26 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

    Dispose of the pelleted beads

18. Quantify 1 µl of eluted sample using a Qubit fluorometer.
19. Important Please first refer to the ligation step below to ensure that the library is diluted to the correct volume.
20. Pool equimolar amounts of each barcoded sample into a 1.5 ml Eppendorf DNA LoBind tube, ensuring that sufficient sample is combined to produce a pooled sample of 700 ng total.
21. Quantify 1 µl of pooled and barcoded DNA using a Qubit fluorometer.
22. Dilute 700 ng pooled sample to 65 µl in nuclease-free water.
23. Optional Action

    If 700 ng of pooled sample exceeds 65 µl in volume, perform an AMPure clean-up with 2.5x Agencourt AMPure XP beads to pooled sample volume, eluting in 65 µl of nuclease-free water.
24. End of Step Take forward the pooled samples into the next step. However, at this point it is also possible to store the sample at 4°C overnight.

1. Adapter Mix II Expansion use

   Protocols that use the Native Barcoding Expansions require 5 μl of AMII per reaction. Native Barcoding Expansions EXP-NBD104/NBD114 contain sufficient AMII for 6 reactions (or 12 reactions when sequencing on Flongle). This assumes that all barcodes are used in one sequencing run.

   The Adapter Mix II expansion provides additional AMII for customers who are running subsets of barcodes, and allows a further 12 reactions (24 on Flongle).

2. Thaw the Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature, mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.
3. Spin down the T4 Ligase and the Adapter Mix II (AMII), and place on ice.
4. Thaw one tube of Long Fragment Buffer (LFB) at room temperature and mix by vortexing, then spin down and place on ice.
5. Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.

   Reagent	Volume
   700 ng pooled barcoded sample	65 µl
   Adapter Mix II (AMII)	5 µl
   NEBNext Quick Ligation Reaction Buffer (5X)	20 µl
   Quick T4 DNA Ligase	10 µl
   Total	100 µl

6. Ensure the components are thoroughly mixed by pipetting, and spin down.
7. Incubate the reaction for 10 minutes at room temperature.
8. Resuspend the AMPure XP beads by vortexing.
9. Add 40 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
10. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
11. Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
12. Wash the beads by adding 250 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
13. Repeat the previous step.
14. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
15. Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at 37°C to improve the recovery of long fragments.
16. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
17. Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

    Dispose of the pelleted beads

18. Quantify 1 µl of adapter ligated and barcoded DNA using a Qubit fluorometer - recovery aim ~430 ng.
19. End of Step The prepared library is used for loading onto the flow cell. Store the library on ice or at 4°C until ready to load.
20. 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, reloading 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.
    For further information, please refer to the DNA library stability Know-How document.

21. Optional Action

    If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

    Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.

1. Tip Priming and loading a flow cell

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

2. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), 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.
3. 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.
4. 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.

   

   

5. 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.

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

   

7. 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.
8. 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.

   

9. 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.

   

10. Thoroughly mix the contents of the Loading Beads (LB) by pipetting.
11. Important The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
12. Tip Using the Loading Beads

    Demo of how to use the Loading Beads.

13. In a new tube, prepare the library for loading as follows:

    Reagent	Volume per flow cell
    Sequencing Buffer (SQB)	37.5 µl
    Loading Beads (LB), mixed immediately before use	25.5 µl
    DNA library	12 µl
    Total	75 µl

    Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer (SQB) and Loading Beads (LB) because the fuel in the buffer will start to be consumed by the adapter.

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. Overview of nanopore data analysis

   For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

2. How to start sequencing

   The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. There are multiple options for how to carry out sequencing:

   1. Data acquisition and basecalling in real-time using MinKNOW on a computer

   Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section.

   2. Data acquisition and basecalling in real-time using the GridION device

   Follow the instructions in the GridION user manual.

   3. Data acquisition and basecalling in real-time using the MinION Mk1C device

   Follow the instructions in the MinION Mk1C user manual.

   4. Data acquisition and basecalling in real-time using the PromethION device

   Follow the instructions in the PromethION user manual or the PromethION 2 Solo user manual.

   5. Data acquisition using MinKNOW on a computer and basecalling at a later time using MinKNOW

   Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section. When setting your experiment parameters, set the Basecalling tab to OFF. After the sequencing experiment has completed, follow the instructions in the Post-run analysis section of the MinKNOW protocol.

1. Additional resources for analysing your ASFV data

   Note: Information subject to change, please refer to No part gets left behind: Tiled nanopore sequencing of whole ASFV genomes stitched together using Lilo by Amanda Warr et al., 2021 for most recent updates.

2. Bioinformatic processing of ASFV genomes sequenced with shotgun sequencing

   The shotgun sequencing data were basecalled and demultiplexed using MinKNOW (v19.06.8) using Fast basecalling.
   Following basecalling the reads were aligned to an ASFV genome using minimap2 to identify ASFV reads, the .fast5 files for these reads were extracted using fast5_subset from the ont_fast5_api and these again using high accuracy basecalling (this reduces basecalling time, suitable for when working with lower spec laptops/computers that have low GPU capacity).
   The reads were assembled with Flye (v2.6) - additional Flye resources available following the link: Assembly of long, error-prone reads using repeat graphs and polished three times with Medaka (v0.7.1).
   Comparisons of quantity of data produced and the proportion of which were ASFV reads were done using NanoComp (v1.28.1) - additional Nanopack resources available following the link: NanoPack: visualizing and processing long-read sequencing data.

3. Bioinformatic processing of ASFV genomes from tiled amplicons with Lilo

   The data were basecalled and demultiplexed using Guppy (v5.0.14) using high or super accuracy model on a GPU.

   The snakemake pipeline Lilo was used, taking the following steps:

   1. Use Porechop (v0.2.3) to remove any sequencing adapters or barcodes that have made it through demultiplexing.
   2. Align to a reference with minimap2 (v2.22) and samtools (v1.12) and separate reads into amplicons by alignment position with bedtools (v2.30.0).
   3. Select reads of the expected amplicon length (+/-5%) and subset to 300X
   4. Select the read with highest average base quality within +/-1% of the median length of reads for the amplicon to be the “reference” using bioawk v1 and remove any amplicons with fewer than 40 reads (targeting the median length allows for flexibility for large insertions or deletions).
   5. Pool amplicon reads and references back into their original non-overlapping pools.
   6. Polish the pools three times with Medaka (v1.4.4) and combine resulting polished amplicons.
   7. Align to the reference with minimap2 and remove soft clipped bases (these likely represent missed barcodes or adapters).
   8. Run porechop to remove primers from the amplicons.
   9. Merge the amplicons with scaffold_builder (v2.3).

   The required input to Lilo are demultiplexed reads in FASTQ format in a directory named “raw/”, a reference FASTA, a .bed file of primer alignments (as output by primal scheme), and a .csv of primer sequences (if there are ambiguous bases it is advised to expand them first) and a config file, described on the GitHub page. It is adaptable to any species (with a single genome fragment/chromosome) with any tiled primer scheme. The pipeline outputs a FASTA file containing the assembled genome.

4. ARTIC assemblies

   A subset of genomes were also assembled using the ARTIC pipeline (v1.2.1) following the bioinformatics SOP using the Medaka method.

5. Quality control of assembled genomes

   Quast (v5.0.2) was used to compare the assembled genomes to the most closely related publicly available ASFV assembly according to BLAST alignment (MN715134.1) - links and additional resources available following the link: Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells.
   Samples where both WGS and tiled sequencing were used were compared for overall structure using nucmer (v4.0.0beta2) - links and aditional reources available following the link: MUMmer4: A fast and versatile genome alignment system.

6. Phylogeny

   The phylogeny analysis was limited to the tiled genomes, as these were the most accurate assemblies, and publicly available genomes. These were aligned using Mafft (v7.467) and maximum likelihood trees constructed using iqtree (v2.0.5).

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. 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 output from PCR

   Observation	Possible cause	Comments and actions
   Low output from PCR	Insufficient primer	Increase the amount of primer pool in the PCR reaction by 0.25-1 µl, decrease water proportionally.
   -	Insufficient DNA template	Use undiluted DNA.
   -	Thermocycler fault	Ensure that you use a thermocycler that has been recently calibrated.
   -	Insufficient viral DNA and an abundance of host DNA	Use a host depletion method such as NEBNext® Microbiome DNA Enrichment Kit before PCR*.
   -	Low representation of a particular primer in the sequencing data	Spike in a small quantity of the primer in the 100 µM pool before future runs

   * This may be prohibitively expensive at scale, but for precious samples this will have a noticeable impact on amplicon yield.

4. 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.