Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096)

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. 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.
3. Introduction to the dual barcoding protocol

   This protocol allows massively parallel sequencing of up to 2,304 samples (gDNA or amplicons) on a single flow cell. It uses both the PCR Barcoding Expansion 1-96 (EXP-PBC096) and the Native Barcoding Expansions 1-12 and 13-24 (EXP-NBD104 and EXP-NBD114). First, up to 96 barcodes are added to the DNA ends with PCR. Up to 24 pools of 96 samples can then have a secondary barcode added through the ligation of one of 24 native barcodes. All primary pools can then be combined into a single library and sequenced.

   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

   Library preparation

   You will need to:
   - Prepare the DNA ends for adapter attachment
   - Attach barcoding adapters supplied in the 96 PCR Barcoding kit to the DNA ends
   - Amplify each barcoded sample by PCR, then pool the samples together
   - Prepare the DNA ends for native barcode attachment
   - Ligate native barcodes to the DNA ends
   - Attach sequencing adapters supplied in the kit to the DNA ends
   - Prime the flow cell, and load your DNA 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
   - Use the Guppy software to split the reads by barcode

4. 96 barcode sequences

   Component	Sequence
   BC01 / RB01	AAGAAAGTTGTCGGTGTCTTTGTG
   BC02 / RB02	TCGATTCCGTTTGTAGTCGTCTGT
   BC03 / RB03	GAGTCTTGTGTCCCAGTTACCAGG
   BC04 / RB04	TTCGGATTCTATCGTGTTTCCCTA
   BC05 / RB05	CTTGTCCAGGGTTTGTGTAACCTT
   BC06 / RB06	TTCTCGCAAAGGCAGAAAGTAGTC
   BC07 / RB07	GTGTTACCGTGGGAATGAATCCTT
   BC08 / RB08	TTCAGGGAACAAACCAAGTTACGT
   BC09 / RB09	AACTAGGCACAGCGAGTCTTGGTT
   BC10 / RB10	AAGCGTTGAAACCTTTGTCCTCTC
   BC11 / RB11	GTTTCATCTATCGGAGGGAATGGA
   BC12 / RB12	CAGGTAGAAAGAAGCAGAATCGGA
   BC13 / 16S13 / RB13	AGAACGACTTCCATACTCGTGTGA
   BC14 / 16S14 / RB14	AACGAGTCTCTTGGGACCCATAGA
   BC15 / 16S15 / RB15	AGGTCTACCTCGCTAACACCACTG
   BC16 / 16S16 / RB16	CGTCAACTGACAGTGGTTCGTACT
   BC17 / 16S17 / RB17	ACCCTCCAGGAAAGTACCTCTGAT
   BC18 / 16S18 / RB18	CCAAACCCAACAACCTAGATAGGC
   BC19 / 16S19 / RB19	GTTCCTCGTGCAGTGTCAAGAGAT
   BC20 / 16S20 / RB20	TTGCGTCCTGTTACGAGAACTCAT
   BC21 / 16S21 / RB21	GAGCCTCTCATTGTCCGTTCTCTA
   BC22 / 16S22 / RB22	ACCACTGCCATGTATCAAAGTACG
   BC23 / 16S23 / RB23	CTTACTACCCAGTGAACCTCCTCG
   BC24 / 16S24 / RB24	GCATAGTTCTGCATGATGGGTTAG
   BC25 / RB25	GTAAGTTGGGTATGCAACGCAATG
   BC26 / RB26	CATACAGCGACTACGCATTCTCAT
   BC27 / RB27	CGACGGTTAGATTCACCTCTTACA
   BC28 / RB28	TGAAACCTAAGAAGGCACCGTATC
   BC29 / RB29	CTAGACACCTTGGGTTGACAGACC
   BC30 / RB30	TCAGTGAGGATCTACTTCGACCCA
   BC31 / RB31	TGCGTACAGCAATCAGTTACATTG
   BC32 / RB32	CCAGTAGAAGTCCGACAACGTCAT
   BC33 / RB33	CAGACTTGGTACGGTTGGGTAACT
   BC34 / RB34	GGACGAAGAACTCAAGTCAAAGGC
   BC35 / RB35	CTACTTACGAAGCTGAGGGACTGC
   BC36 / RB36	ATGTCCCAGTTAGAGGAGGAAACA
   BC37 / RB37	GCTTGCGATTGATGCTTAGTATCA
   BC38 / RB38	ACCACAGGAGGACGATACAGAGAA
   BC39 / RB39	CCACAGTGTCAACTAGAGCCTCTC
   BC40 / RB40	TAGTTTGGATGACCAAGGATAGCC
   BC41 / RB41	GGAGTTCGTCCAGAGAAGTACACG
   BC42 / RB42	CTACGTGTAAGGCATACCTGCCAG
   BC43 / RB43	CTTTCGTTGTTGACTCGACGGTAG
   BC44 / RB44	AGTAGAAAGGGTTCCTTCCCACTC
   BC45 / RB45	GATCCAACAGAGATGCCTTCAGTG
   BC46 / RB46	GCTGTGTTCCACTTCATTCTCCTG
   BC47 / RB47	GTGCAACTTTCCCACAGGTAGTTC
   BC48 / RB48	CATCTGGAACGTGGTACACCTGTA
   BC49 / RB49	ACTGGTGCAGCTTTGAACATCTAG
   BC50 / RB50	ATGGACTTTGGTAACTTCCTGCGT
   BC51 / RB51	GTTGAATGAGCCTACTGGGTCCTC
   BC52 / RB52	TGAGAGACAAGATTGTTCGTGGAC
   BC53 / RB53	AGATTCAGACCGTCTCATGCAAAG
   BC54 / RB54	CAAGAGCTTTGACTAAGGAGCATG
   BC55 / RB55	TGGAAGATGAGACCCTGATCTACG
   BC56 / RB56	TCACTACTCAACAGGTGGCATGAA
   BC57 / RB57	GCTAGGTCAATCTCCTTCGGAAGT
   BC58 / RB58	CAGGTTACTCCTCCGTGAGTCTGA
   BC59 / RB59	TCAATCAAGAAGGGAAAGCAAGGT
   BC60 / RB60	CATGTTCAACCAAGGCTTCTATGG
   BC61 / RB61	AGAGGGTACTATGTGCCTCAGCAC
   BC62 / RB62	CACCCACACTTACTTCAGGACGTA
   BC63 / RB63	TTCTGAAGTTCCTGGGTCTTGAAC
   BC64 / RB64	GACAGACACCGTTCATCGACTTTC
   BC65 / RB65	TTCTCAGTCTTCCTCCAGACAAGG
   BC66 / RB66	CCGATCCTTGTGGCTTCTAACTTC
   BC67 / RB67	GTTTGTCATACTCGTGTGCTCACC
   BC68 / RB68	GAATCTAAGCAAACACGAAGGTGG
   BC69 / RB69	TACAGTCCGAGCCTCATGTGATCT
   BC70 / RB70	ACCGAGATCCTACGAATGGAGTGT
   BC71 / RB71	CCTGGGAGCATCAGGTAGTAACAG
   BC72 / RB72	TAGCTGACTGTCTTCCATACCGAC
   BC73 / RB73	AAGAAACAGGATGACAGAACCCTC
   BC74 / RB74	TACAAGCATCCCAACACTTCCACT
   BC75 / RB75	GACCATTGTGATGAACCCTGTTGT
   BC76 / RB76	ATGCTTGTTACATCAACCCTGGAC
   BC77 / RB77	CGACCTGTTTCTCAGGGATACAAC
   BC78 / RB78	AACAACCGAACCTTTGAATCAGAA
   BC79 / RB79	TCTCGGAGATAGTTCTCACTGCTG
   BC80 / RB80	CGGATGAACATAGGATAGCGATTC
   BC81 / RB81	CCTCATCTTGTGAAGTTGTTTCGG
   BC82 / RB82	ACGGTATGTCGAGTTCCAGGACTA
   BC83 / RB83	TGGCTTGATCTAGGTAAGGTCGAA
   BC84 / RB84	GTAGTGGACCTAGAACCTGTGCCA
   BC85 / RB85	AACGGAGGAGTTAGTTGGATGATC
   BC86 / RB86	AGGTGATCCCAACAAGCGTAAGTA
   BC87 / RB87	TACATGCTCCTGTTGTTAGGGAGG
   BC88 / RB88	TCTTCTACTACCGATCCGAAGCAG
   BC89 / RB89	ACAGCATCAATGTTTGGCTAGTTG
   BC90 / RB90	GATGTAGAGGGTACGGTTTGAGGC
   BC91 / RB91	GGCTCCATAGGAACTCACGCTACT
   BC92 / RB92	TTGTGAGTGGAAAGATACAGGACC
   BC93 / RB93	AGTTTCCATCACTTCAGACTTGGG
   BC94 / RB94	GATTGTCCTCAAACTGCCACCTAC
   BC95 / RB95	CCTGTCTGGAAGAAGAATGGACTT
   BC96 / RB96	CTGAACGGTCATAGAGTCCACCAT

5. Native barcode sequences

   Below is the full list of our native barcode (NB01-96) sequences. The first 24 unique barcodes are available in the Native Barcoding Kit 24 V14 (SQK-NBD114.24). The Native Barcoding Kit 96 V14 (SQK-NBD114.96) include the first 24 native barcodes, with the additional 72 unique barcodes. The native barcodes are shipped at 640 nM.

   In addition to the barcodes, there are also flanking sequences which add an extra level of context during analysis.

   Barcode flanking sequences:

   Forward sequence: 5' - AAGGTTAA - barcode - CAGCACCT - 3'
   Reverse sequence: 5' - GGTGCTG - barcode - TTAACCTTAGCAAT - 3'

   
   

   Native barcode sequences

   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
   NB25	GTAAGTTGGGTATGCAACGCAATG	CATTGCGTTGCATACCCAACTTAC
   NB26	CATACAGCGACTACGCATTCTCAT	ATGAGAATGCGTAGTCGCTGTATG
   NB27	CGACGGTTAGATTCACCTCTTACA	TGTAAGAGGTGAATCTAACCGTCG
   NB28	TGAAACCTAAGAAGGCACCGTATC	GATACGGTGCCTTCTTAGGTTTCA
   NB29	CTAGACACCTTGGGTTGACAGACC	GGTCTGTCAACCCAAGGTGTCTAG
   NB30	TCAGTGAGGATCTACTTCGACCCA	TGGGTCGAAGTAGATCCTCACTGA
   NB31	TGCGTACAGCAATCAGTTACATTG	CAATGTAACTGATTGCTGTACGCA
   NB32	CCAGTAGAAGTCCGACAACGTCAT	ATGACGTTGTCGGACTTCTACTGG
   NB33	CAGACTTGGTACGGTTGGGTAACT	AGTTACCCAACCGTACCAAGTCTG
   NB34	GGACGAAGAACTCAAGTCAAAGGC	GCCTTTGACTTGAGTTCTTCGTCC
   NB35	CTACTTACGAAGCTGAGGGACTGC	GCAGTCCCTCAGCTTCGTAAGTAG
   NB36	ATGTCCCAGTTAGAGGAGGAAACA	TGTTTCCTCCTCTAACTGGGACAT
   NB37	GCTTGCGATTGATGCTTAGTATCA	TGATACTAAGCATCAATCGCAAGC
   NB38	ACCACAGGAGGACGATACAGAGAA	TTCTCTGTATCGTCCTCCTGTGGT
   NB39	CCACAGTGTCAACTAGAGCCTCTC	GAGAGGCTCTAGTTGACACTGTGG
   NB40	TAGTTTGGATGACCAAGGATAGCC	GGCTATCCTTGGTCATCCAAACTA
   NB41	GGAGTTCGTCCAGAGAAGTACACG	CGTGTACTTCTCTGGACGAACTCC
   NB42	CTACGTGTAAGGCATACCTGCCAG	CTGGCAGGTATGCCTTACACGTAG
   NB43	CTTTCGTTGTTGACTCGACGGTAG	CTACCGTCGAGTCAACAACGAAAG
   NB44	AGTAGAAAGGGTTCCTTCCCACTC	GAGTGGGAAGGAACCCTTTCTACT
   NB45	GATCCAACAGAGATGCCTTCAGTG	CACTGAAGGCATCTCTGTTGGATC
   NB46	GCTGTGTTCCACTTCATTCTCCTG	CAGGAGAATGAAGTGGAACACAGC
   NB47	GTGCAACTTTCCCACAGGTAGTTC	GAACTACCTGTGGGAAAGTTGCAC
   NB48	CATCTGGAACGTGGTACACCTGTA	TACAGGTGTACCACGTTCCAGATG
   NB49	ACTGGTGCAGCTTTGAACATCTAG	CTAGATGTTCAAAGCTGCACCAGT
   NB50	ATGGACTTTGGTAACTTCCTGCGT	ACGCAGGAAGTTACCAAAGTCCAT
   NB51	GTTGAATGAGCCTACTGGGTCCTC	GAGGACCCAGTAGGCTCATTCAAC
   NB52	TGAGAGACAAGATTGTTCGTGGAC	GTCCACGAACAATCTTGTCTCTCA
   NB53	AGATTCAGACCGTCTCATGCAAAG	CTTTGCATGAGACGGTCTGAATCT
   NB54	CAAGAGCTTTGACTAAGGAGCATG	CATGCTCCTTAGTCAAAGCTCTTG
   NB55	TGGAAGATGAGACCCTGATCTACG	CGTAGATCAGGGTCTCATCTTCCA
   NB56	TCACTACTCAACAGGTGGCATGAA	TTCATGCCACCTGTTGAGTAGTGA
   NB57	GCTAGGTCAATCTCCTTCGGAAGT	ACTTCCGAAGGAGATTGACCTAGC
   NB58	CAGGTTACTCCTCCGTGAGTCTGA	TCAGACTCACGGAGGAGTAACCTG
   NB59	TCAATCAAGAAGGGAAAGCAAGGT	ACCTTGCTTTCCCTTCTTGATTGA
   NB60	CATGTTCAACCAAGGCTTCTATGG	CCATAGAAGCCTTGGTTGAACATG
   NB61	AGAGGGTACTATGTGCCTCAGCAC	GTGCTGAGGCACATAGTACCCTCT
   NB62	CACCCACACTTACTTCAGGACGTA	TACGTCCTGAAGTAAGTGTGGGTG
   NB63	TTCTGAAGTTCCTGGGTCTTGAAC	GTTCAAGACCCAGGAACTTCAGAA
   NB64	GACAGACACCGTTCATCGACTTTC	GAAAGTCGATGAACGGTGTCTGTC
   NB65	TTCTCAGTCTTCCTCCAGACAAGG	CCTTGTCTGGAGGAAGACTGAGAA
   NB66	CCGATCCTTGTGGCTTCTAACTTC	GAAGTTAGAAGCCACAAGGATCGG
   NB67	GTTTGTCATACTCGTGTGCTCACC	GGTGAGCACACGAGTATGACAAAC
   NB68	GAATCTAAGCAAACACGAAGGTGG	CCACCTTCGTGTTTGCTTAGATTC
   NB69	TACAGTCCGAGCCTCATGTGATCT	AGATCACATGAGGCTCGGACTGTA
   NB70	ACCGAGATCCTACGAATGGAGTGT	ACACTCCATTCGTAGGATCTCGGT
   NB71	CCTGGGAGCATCAGGTAGTAACAG	CTGTTACTACCTGATGCTCCCAGG
   NB72	TAGCTGACTGTCTTCCATACCGAC	GTCGGTATGGAAGACAGTCAGCTA
   NB73	AAGAAACAGGATGACAGAACCCTC	GAGGGTTCTGTCATCCTGTTTCTT
   NB74	TACAAGCATCCCAACACTTCCACT	AGTGGAAGTGTTGGGATGCTTGTA
   NB75	GACCATTGTGATGAACCCTGTTGT	ACAACAGGGTTCATCACAATGGTC
   NB76	ATGCTTGTTACATCAACCCTGGAC	GTCCAGGGTTGATGTAACAAGCAT
   NB77	CGACCTGTTTCTCAGGGATACAAC	GTTGTATCCCTGAGAAACAGGTCG
   NB78	AACAACCGAACCTTTGAATCAGAA	TTCTGATTCAAAGGTTCGGTTGTT
   NB79	TCTCGGAGATAGTTCTCACTGCTG	CAGCAGTGAGAACTATCTCCGAGA
   NB80	CGGATGAACATAGGATAGCGATTC	GAATCGCTATCCTATGTTCATCCG
   NB81	CCTCATCTTGTGAAGTTGTTTCGG	CCGAAACAACTTCACAAGATGAGG
   NB82	ACGGTATGTCGAGTTCCAGGACTA	TAGTCCTGGAACTCGACATACCGT
   NB83	TGGCTTGATCTAGGTAAGGTCGAA	TTCGACCTTACCTAGATCAAGCCA
   NB84	GTAGTGGACCTAGAACCTGTGCCA	TGGCACAGGTTCTAGGTCCACTAC
   NB85	AACGGAGGAGTTAGTTGGATGATC	GATCATCCAACTAACTCCTCCGTT
   NB86	AGGTGATCCCAACAAGCGTAAGTA	TACTTACGCTTGTTGGGATCACCT
   NB87	TACATGCTCCTGTTGTTAGGGAGG	CCTCCCTAACAACAGGAGCATGTA
   NB88	TCTTCTACTACCGATCCGAAGCAG	CTGCTTCGGATCGGTAGTAGAAGA
   NB89	ACAGCATCAATGTTTGGCTAGTTG	CAACTAGCCAAACATTGATGCTGT
   NB90	GATGTAGAGGGTACGGTTTGAGGC	GCCTCAAACCGTACCCTCTACATC
   NB91	GGCTCCATAGGAACTCACGCTACT	AGTAGCGTGAGTTCCTATGGAGCC
   NB92	TTGTGAGTGGAAAGATACAGGACC	GGTCCTGTATCTTTCCACTCACAA
   NB93	AGTTTCCATCACTTCAGACTTGGG	CCCAAGTCTGAAGTGATGGAAACT
   NB94	GATTGTCCTCAAACTGCCACCTAC	GTAGGTGGCAGTTTGAGGACAATC
   NB95	CCTGTCTGGAAGAAGAATGGACTT	AAGTCCATTCTTCTTCCAGACAGG
   NB96	CTGAACGGTCATAGAGTCCACCAT	ATGGTGGACTCTATGACCGTTCAG

6. Important We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.
7. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • PCR Barcoding Expansion Pack 1-96 (EXP-PBC096)
   • Native Barcoding Expansion Pack 1-12 and/or 13-24 (EXP-NBD104/EXP-NBD114)
   • Ligation Sequencing Kit 1D (SQK-LSK109)
   • FLO-MIN106 (R9.4.1) flow cells
   • Flow Cell Wash Kit (EXP-WSH004)

1. For this protocol, you will need the following amounts of each DNA sample to be barcoded in 45 µl:
   • 1 µg (or 100-200 fmol) gDNA is required for R9.4.1 flow cells
   • 1.5-3 µg (or 150-300 fmol) gDNA is required for R10.3 flow cells
2. Input DNA

   How to QC your input DNA

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

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

   Chemical contaminants

   Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

3. PCR Barcoding Expansion Pack 1-96 (EXP-PBC096)

   The kit allows up to 96 different libraries to be combined; each of these libraries will be barcoded with one of the 96 PCR barcodes (BC1-BC96). These libraries can then be barcoded for the second time with a native barcode from the Native Barcoding Expansions.

   The reagents are divided between two 96 tube plates. One plate (blue caps) contains barcode adapters that are ligated to the DNA. All tubes in this plate have identical content. The other plate (white caps) contains the barcodes, one barcode per tube.

   

   Name	No. of plates	Fill volume per well (µl)
   PCR Primer mix	1	24
   Barcode adapter plate	1	240

4. Layout of barcodes in the 96 tube plate

   The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.

   

5. 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

6. Ligation Sequencing Kit contents (SQK-LSK109)

   

   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

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

   

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

8. Adapter Mix II Expansion contents (EXP-AMII001)

   

   Name	Acronym	Cap colour	No. of tubes	Fill volume per vial (μl)
   Adapter Mix II	AMII	Green	2	40

9. 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 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 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).

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. 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. Currently, the FASTQ Barcoding workflow in EPI2ME is not compatible with dual-barcoded reads.
   EPI2ME installation and use
   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. Important Optional fragmentation and size selection

   By default, the protocol contains no DNA fragmentation step, however in some cases it may be advantageous to fragment your sample. For example, when working with lower amounts of input gDNA (100 ng – 500 ng), fragmentation will increase the number of DNA molecules and therefore increase throughput. Instructions are available in the DNA Fragmentation section of Extraction methods.

   Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.

2. Prepare the DNA in nuclease-free water.
   • Transfer 100–200 fmol DNA for each sample to be barcoded into a separate 1.5 ml Eppendorf DNA LoBind tube
   • Adjust the volume to 45 μl with nuclease-free water
   • Mix thoroughly by flicking the tube to avoid unwanted shearing
   • Spin down briefly in a microfuge

3. In a 0.2 ml 96 well PCR plate, set up the end-repair / dA-tailing reactions as follows:

   Reagent	Volume
   DNA sample	45 µl
   Ultra II End-prep reaction buffer	7 µl
   Ultra II End-prep enzyme mix	3 µl
   Nuclease-free water	5 µl
   Total	60 µl

4. Mix by pipetting.
5. Seal the plate with adhesive film or PCR strip caps, spin down in a centrifuge and incubate for 5 minutes at 20 °C and 5 minutes at 65 °C using the thermal cycler.

   If condensation is observed in the plate after the thermocycling, briefly spin down the plate contents in a centrifuge.

6. Resuspend the AMPure XP beads by vortexing.
7. Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
8. Allow DNA to bind to beads for 5 minutes at room temperature.
9. Prepare sufficient fresh 70% ethanol in nuclease-free water.
10. Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
11. Keep the tube on the magnet and wash the beads with 180 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
12. Repeat the previous step.
13. Cover the plate with adhesive film and leave plate on magnet for 2 minutes to allow residual liquid to collect at the bottom. Remove the adhesive film, return the plate to the magnet and aspirate residual wash solution.
14. Briefly incubate the plate on a thermal cycler at 37° C with the lid open and the plate wells unsealed.
15. Remove the plate from the magnet and resuspend pellet in 31 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 eluate once it is clear and colourless. Transfer each eluted sample to a new 96-well PCR plate.
18. Quantify 1 µl of end-prepped DNA using a Qubit fluorometer - recovery aim 70–140 fmol.
19. End of Step Take forward approximately 70–140 fmol of end-prepped DNA in 30 µl nuclease-free water into adapter ligation.

1. Add the reagents to a fresh 96-well plate, in the order given below:

   Between each addition, pipette mix 10-20 times.

   Reagent	Volume
   End-prepped DNA	30 µl
   Barcode Adapter	20 µl
   Blunt/TA Ligase Master Mix	50 µl
   Total	100 µl

2. Mix by pipetting.
3. Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.
4. Incubate the reaction for 10 minutes at room temperature.
5. Resuspend the AMPure XP beads by vortexing.
6. Add 40 µl of resuspended AMPure XP beads to each sample and mix by pipetting up and down ten times.
7. Allow DNA to bind to beads for 5 minutes at room temperature.
8. Prepare sufficient fresh 70% ethanol in nuclease-free water.
9. Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
10. Keep the tube on the magnet and wash the beads with 180 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
11. Repeat the previous step.
12. Cover the plate with adhesive film and leave plate on magnet for 2 minutes to allow residual liquid to collect at the bottom. Remove the adhesive film, return the plate to the magnet and aspirate residual wash solution.
13. Briefly incubate the plate on a thermal cycler at 37° C with the lid open and the plate wells unsealed.
14. Remove the plate from the magnet and resuspend pellet in 25 µl nuclease-free water. Incubate for 2 minutes at room temperature.
15. Pellet the beads on a magnet until the eluate is clear and colourless.
16. Remove eluate once it is clear and colourless. Transfer each eluted sample to a new 96-well PCR plate.
17. Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.
18. Dilute the library to 20–30 fmol with nuclease-free water or 10 mM Tris-HCl pH 8.5.

1. Capping and decapping the 96 well format

   

2. Layout of barcodes in the 96 tube plate

   The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.

   

3. Set up a barcoding PCR reaction as follows for each library:

   The following is written for LongAmp Taq, but can be adapted for use with other polymerases.

   Between each addition, pipette mix 10-20 times.

   Reagent	Volume
   PCR Barcode (one of BC1-BC96, at 10 µM)	1 µl
   Adapter-ligated DNA	2 µl
   LongAmp Taq 2x master mix	25 µl
   Nuclease-free water	22 µl
   Total volume	50 µl

4. The amount of input DNA may need to be adjusted depending on application. For example, for sequencing human or larger genomes, we recommend putting ~50 ng DNA into a PCR reaction. For amplicons or smaller genomes, the 20 ng stated above is sufficient. If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.
5. Mix by pipetting.
6. Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.
7. Amplify using the following cycling conditions:

   Cycle step	Temperature	Time	No. of cycles
   Initial denaturation	95 °C	3 mins	1
   Denaturation	95 °C	15 secs	15-18 (b)
   Annealing	62 °C (a)	15 secs (a)	15-18 (b)
   Extension	65 °C (c)	dependent on length of target fragment (d)	15-18 (b)
   Final extension	65 °C	dependent on length of target fragment (d)	1
   Hold	4 °C	∞

   a. This is specific to the Oxford Nanopore primer and should be maintained

   b. Adjust accordingly if input quantities are altered

   c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

   d. Adjust accordingly for different lengths of amplicons and the type of polymerase that is being used (LongAmp Taq amplifies at a rate of 50 seconds per kb)

8. Resuspend the AMPure XP beads by vortexing.
9. Add 35 µl of of resuspended AMPure XP beads to each sample and mix by pipetting the entire combined volume up and down 10 times.
10. Incubate for 5 minutes at room temperature.
11. Prepare sufficient fresh 70% ethanol in nuclease-free water.
12. Pellet the beads on a magnet for at least 2 min, or until the supernatant is clear. Keep the plate on the magnet and pipette off the supernatant.
13. Wash each pellet of beads by adding 200 µl of freshly-prepared 70% ethanol. Resuspend each pellet thoroughly by pipetting the entire volume of buffer up and down ten times. Return the plate to the magnetic rack and allow the beads to pellet until the supernatant is clear. Remove the supernatant using a pipette and discard.
14. Repeat the previous step.
15. Seal the plate. Spin down and place the plate back on the magnet. Pipette off any residual supernatant.
16. Remove the plate from the magnetic rack and resuspend each pellet in 21 µl nuclease-free water, pipetting the entire volume up and down 10 times. 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 21 µl of each eluate into a well of a clean 96-well plate.
19. Quantify 1 µl of each barcoded DNA sample using a Qubit fluorometer.
20. Optional Action

    If your DNA samples are of mixed sizes, we recommend either running a subset of samples on an Agilent Bioanalyzer, or estimating the DNA size from a gel.
21. Using the DNA mass (calculated using the Qubit fluorometer) and size distribution (calculated using a gel or Agilent Bioanalyzer), pool equimolar quantities of barcoded amplicons in batches of 96, ensuring that every sample within a given pool has a unique barcode.

    Using the DNA mass (calculated using the Qubit fluorometer) and size distribution (calculated using a gel or Agilent Bioanalyzer), pool equimolar quantities of barcoded amplicons in batches of 96 or fewer, depending on how many of the 96 barcodes were used. Ensure that every sample within a given pool has a unique barcode.

22. Important Note that after the subsequent end-prep step, you will need 100–200 fmol of DNA for each pool of samples to take into the native barcode ligation step.

1. Mix the following reagents in a separate 0.2 ml thin-walled PCR tube for each pool of samples:

   Reagent	Volume
   Pool of barcoded DNA samples	20 µl
   Ultra II End-prep reaction buffer	7 µl
   Ultra II End-prep enzyme mix	3 µl
   Nuclease-free water	30 µl
   Total	60 µl

2. Mix by pipetting.
3. Using a thermal cycler, incubate at 20° C for 15 minutes and 65° C for 5 mins.
4. Transfer each sample to a separate 1.5 ml Eppendorf DNA LoBind tube.
5. Resuspend the AMPure XP beads by vortexing.
6. Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
7. Allow DNA to bind to beads for 5 minutes at room temperature.
8. Prepare sufficient fresh 70% ethanol in nuclease-free water.
9. Keep the tube on the magnet and wash the beads with 180 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
10. 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.
11. Repeat the previous step.
12. 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.
13. Remove the tube from the magnetic rack and resuspend the pellet in 23.5 µl nuclease-free water. Incubate for 2 minutes at room temperature.
14. Pellet the beads on a magnet until the eluate is clear and colourless.
15. Remove and retain 23.5 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
16. Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.
17. End of Step Take forward 100–200 fmol of end-prepped DNA in 22.5 µl into native barcode ligation.

1. 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.
2. 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.
3. Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:

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

4. Mix well by pipetting and spin down.
5. Incubate the reaction for 10 minutes at room temperature.
6. Resuspend the AMPure XP beads by vortexing.
7. Add 20 µl of resuspended AMPure XP beads to the reaction and mix by gently flicking the tube.
8. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
9. Prepare sufficient fresh 70% ethanol in nuclease-free water.
10. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
11. 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.
12. Repeat the previous step.
13. 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.
14. Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water. Incubate for 2 minutes at room temperature.
15. Pellet the beads on a magnet until the eluate is clear and colourless.
16. Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
17. Quantify 1 µl of eluted sample using a Qubit fluorometer.
18. Analyse 1 µl of sample using the Agilent Bioanalyzer. Determine the average amplicon size from this data, and use this to calculate the input sample volume for the next step.
19. Pool equimolar amounts of each barcoded sample into a 1.5 ml Eppendord DNA LoBind tube, ensuring that sufficient sample is combined to produce a pooled sample of 0.2 pmol total.
20. Quantify 1 µl of pooled and barcoded DNA using a Qubit fluorometer.
21. Dilute 100–200 fmol pooled sample to 65 µl in nuclease-free water.

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 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 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. Important Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally.
   • To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB)
   • To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)
3. 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.
4. Spin down the T4 Ligase and the Adapter Mix II (AMII), and place on ice.
5. To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.
6. To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
7. Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.

   Reagent	Volume
   100–200 fmol 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

8. Ensure the components are thoroughly mixed by pipetting, and spin down.
9. Incubate the reaction for 10 minutes at room temperature.
10. Resuspend the AMPure XP beads by vortexing.
11. Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
12. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
13. Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
14. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB). 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.
15. Repeat the previous step.
16. Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
17. 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 room temperature. For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.
18. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
19. 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

20. Quantify 1 µl of adapter ligated DNA using a Qubit fluorometer - recovery aim 50–100 fmol.
21. Important We recommend loading 5-50 fmol of the final prepared library onto a flow cell.

    Loading more than the maximal recommended amount of DNA can have a detrimental effect on output as higher quantities of DNA results in a larger number of ligated DNA ends with loaded motor protein. This depletes fuel in the Sequencing Buffer, regardless of whether or not the DNA fragments are being sequenced. This leads to fuel depletion and speed drop-off early in the sequencing run. Dilute the libraries in Elution Buffer if required.

    If you are using the Flongle for sample prep development, we recommend loading 3-20 fmol instead.

22. 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.
23. 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.

24. 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. 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.

13. 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.

    

    

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

    

16. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.

    

    

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.

3. Important Settings in MinKNOW

   When setting up your sequencing experiment in MinKNOW, select the SQK-LSK109 kit only (no Barcoding Expansion Packs) in Kit Selection:
   

1. Barcode demultiplexing in Guppy

   To demultiplex your reads by barcode, use the dual barcoding configuration file, specifying the barcode kit as "EXP-DUAL00":

   
guppy_barcoder --input_path <folder containing FASTQ and/or FASTA files> --save_path <output folder> --config configuration_dual.cfg --barcode_kits "EXP-DUAL00"


   For more information and instructions on how to use Guppy, please refer to the relevant sections of the protocol:
   Barcode demultiplexing
   Quick Start

2. Other data analysis options

   1. EPI2ME platform

   The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment, and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret report with the results. For instructions on how to run an analysis workflow in EPI2ME, please follow the instructions in the EPI2ME protocol, beginning at the "Starting an EPI2ME workflow" step. Please note that the FASTQ Barcoding workflow in EPI2ME is currently not compatible with dual-barcoded reads.

   2. Bioinformatics tutorials

   For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

   3. 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.

   4. 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 Community-developed data analysis tool library. 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. 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 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.