Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096) (DBC_9098_v109_revI_16Apr2020)


1. Overview of the protocol

IMPORTANTE

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.

IMPORTANTE

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.

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

Dual barcoding workflow

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

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

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
IMPORTANTE

We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.

IMPORTANTE

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)

2. Equipment and consumables

Material
  • 100–200 fmol of each DNA sample to be barcoded in 45 µl
  • PCR Barcoding Expansion 1-96 (EXP-PBC096)
  • Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more than 12 samples
  • Ligation Sequencing Kit (SQK-LSK109)
  • Flow Cell Priming Kit (EXP-FLP002)
  • Adapter Mix II Expansion (EXP-AMII001)

Consumibles
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • NEB Blunt/TA Ligase Master Mix (NEB, M0367)
  • NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
  • NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Tubos de PCR de pared fina (0,2 ml)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)

Instrumental
  • Mezclador Hula (mezclador giratorio suave)
  • Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
  • Gradilla magnética
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Microcentrífuga
  • Mezclador vórtex
  • Termociclador
  • Pipeta y puntas P1000
  • Pipeta y puntas P200
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • Pipeta y puntas P2
  • Pipeta multicanal y puntas de varios tamaños
  • Cubeta con hielo
  • Temporizador
Equipo opcional
  • Bioanalizador Agilent (o equivalente)
  • Fluorímetro Qubit (o equivalente para el control de calidad)
  • Centrifugadora Eppendorf 5424 (o equivalente)

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

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.

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.

2655 10999

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

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.

Barcode 96 plate

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

EXP-NBD104 kit 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** ![EXP-NBD114 kit contents](https://a.storyblok.com/f/196663/46251fa9bb/exp-nbd114_kit_contents-355iypje5ymq4ook6maywi-ebb06336aa81351f28d1bc46a1d968f4.svg)
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

Ligation Sequencing Kit contents (SQK-LSK109)

SQK-LSK109 v1

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

Flow Cell Priming Kit contents (EXP-FLP002)

FLP

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

Adapter Mix II Expansion contents (EXP-AMII001)

EXP-AMII001 kit contents

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

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).

3. Computer requirements and software

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. For more information, refer to the MinION Mk1B IT requirements document.

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.

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

4. End-prep

Material
  • 100–200 fmol of each DNA sample to be barcoded in 45 µl

Consumibles
  • NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 0.2 ml 96-well PCR plate

Instrumental
  • Termociclador
  • Cubeta con hielo
  • Centrifuge capable of taking 96-well plates
  • Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
Equipo opcional
  • Fluorímetro Qubit (o equivalente para el control de calidad)
IMPORTANTE

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.

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

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

Mix by pipetting.

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.

Resuspend the AMPure XP beads by vortexing.

Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.

Allow DNA to bind to beads for 5 minutes at room temperature.

Prepare sufficient fresh 70% ethanol in nuclease-free water.

Place on a magnetic rack, allow beads to pellet and pipette off supernatant.

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.

Repeat the previous step.

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.

Briefly incubate the plate on a thermal cycler at 37° C with the lid open and the plate wells unsealed.

Remove the plate from the magnet and resuspend pellet in 31 µl nuclease-free water. Incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove eluate once it is clear and colourless. Transfer each eluted sample to a new 96-well PCR plate.

Quantify 1 µl of end-prepped DNA using a Qubit fluorometer - recovery aim 70–140 fmol.

FIN DEL PROCESO

Take forward approximately 70–140 fmol of end-prepped DNA in 30 µl nuclease-free water into adapter ligation.

5. Ligation of Barcode Adapter

Material
  • Barcode Adapter (BCA)

Consumibles
  • NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 10 mM Tris-HCl pH 8.5
  • 0.2 ml 96-well PCR plate

Instrumental
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Mezclador Hula (mezclador giratorio suave)
  • Mezclador vórtex
  • Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
  • Cubeta con hielo
  • Pipeta multicanal y puntas de varios tamaños
Equipo opcional
  • Bioanalizador Agilent (o equivalente)

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

Mix by pipetting.

Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP beads by vortexing.

Add 40 µl of resuspended AMPure XP beads to each sample and mix by pipetting up and down ten times.

Allow DNA to bind to beads for 5 minutes at room temperature.

Prepare sufficient fresh 70% ethanol in nuclease-free water.

Place on a magnetic rack, allow beads to pellet and pipette off supernatant.

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.

Repeat the previous step.

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.

Briefly incubate the plate on a thermal cycler at 37° C with the lid open and the plate wells unsealed.

Remove the plate from the magnet and resuspend pellet in 25 µl nuclease-free water. Incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove eluate once it is clear and colourless. Transfer each eluted sample to a new 96-well PCR plate.

Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.

Dilute the library to 20–30 fmol with nuclease-free water or 10 mM Tris-HCl pH 8.5.

6. Barcoding PCR

Material
  • PCR Barcodes (BC01-96, at 10 µM)

Consumibles
  • LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • 0.2 ml 96-well PCR plate
  • Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental
  • Termociclador
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
Equipo opcional
  • Fluorímetro Qubit (o equivalente para el control de calidad)

Capping and decapping the 96 well format

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.

Barcode 96 plate

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

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.

Mix by pipetting.

Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.

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)

Resuspend the AMPure XP beads by vortexing.

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.

Incubate for 5 minutes at room temperature.

Prepare sufficient fresh 70% ethanol in nuclease-free water.

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.

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.

Repeat the previous step.

Seal the plate. Spin down and place the plate back on the magnet. Pipette off any residual supernatant.

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.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 21 µl of each eluate into a well of a clean 96-well plate.

Quantify 1 µl of each barcoded DNA sample using a Qubit fluorometer.

MEDIDA OPCIONAL

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.

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.

IMPORTANTE

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.

7. End-prep

Material
  • Up to 24 pools of barcoded amplicons (with up to 96 samples in each pool), in 20 µl

Consumibles
  • NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Tubos de PCR de pared fina (0,2 ml)

Instrumental
  • Termociclador
  • Cubeta con hielo
  • Centrifuge capable of taking 96-well plates
  • Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
Equipo opcional
  • Fluorímetro Qubit (o equivalente para el control de calidad)

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

Mix by pipetting.

Using a thermal cycler, incubate at 20° C for 15 minutes and 65° C for 5 mins.

Transfer each sample to a separate 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the AMPure XP beads by vortexing.

Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.

Allow DNA to bind to beads for 5 minutes at room temperature.

Prepare sufficient fresh 70% ethanol in nuclease-free water.

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.

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.

Repeat the previous step.

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.

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.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 23.5 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.

FIN DEL PROCESO

Take forward 100–200 fmol of end-prepped DNA in 22.5 µl into native barcode ligation.

8. Native barcode ligation

Material
  • Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more than 12 samples

Consumibles
  • Freshly prepared 70% ethanol in nuclease-free water
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • NEB Blunt/TA Ligase Master Mix (NEB, M0367)

Instrumental
  • Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
  • Mezclador Hula (mezclador giratorio suave)
  • Mezclador vórtex
  • Cubeta con hielo
  • Microcentrífuga
  • Pipeta y puntas P1000
  • Pipeta y puntas P100
  • Pipeta y puntas P10
Equipo opcional
  • Fluorímetro Qubit (o equivalente para el control de calidad)

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.

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.

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

Mix well by pipetting and spin down.

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP beads by vortexing.

Add 20 µl of resuspended AMPure XP beads to the reaction and mix by gently flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Prepare sufficient fresh 70% ethanol in nuclease-free water.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.

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.

Repeat the previous step.

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.

Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water. Incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

VERIFICACIÓN

Quantify 1 µl of eluted sample using a Qubit fluorometer.

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.

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.

Quantify 1 µl of pooled and barcoded DNA using a Qubit fluorometer.

Dilute 100–200 fmol pooled sample to 65 µl in nuclease-free water.

9. Adapter ligation and clean-up

Material
  • Long Fragment Buffer (LFB) (tampón para fragmentos largos)
  • Short Fragment Buffer (SFB) (tampón para fragmentos cortos)
  • Elution Buffer (EB) (tampón de elución) del kit de Oxford Nanopore
  • Adapter Mix II (AMII)

Consumibles
  • NEBNext® Quick Ligation Module (NEB, E6056)
  • NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental
  • Microcentrífuga
  • Gradilla magnética
  • Mezclador vórtex
  • Mezclador Hula (mezclador giratorio suave)
Equipo opcional
  • Fluorímetro Qubit (o equivalente para el control de calidad)

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).

IMPORTANTE

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)

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.

Spin down the T4 Ligase and the Adapter Mix II (AMII), and place on ice.

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.

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.

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

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

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP beads by vortexing.

Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

Place on a magnetic rack, allow beads to pellet and pipette off supernatant.

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.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant.

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.

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

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

Quantify 1 µl of adapter ligated DNA using a Qubit fluorometer - recovery aim 50–100 fmol.

IMPORTANTE

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.

FIN DEL PROCESO

The prepared library is used for loading onto the flow cell. Store the library on ice or at 4°C until ready to load.

CONSEJO

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.

MEDIDA OPCIONAL

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.

10. Priming and loading the SpotON flow cell

Material
  • Flow Cell Priming Kit (EXP-FLP002)
  • Loading Beads (LB)
  • Sequencing Buffer (SQB)

Consumibles
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)

Instrumental
  • MinION Mk1B or Mk1C
  • SpotON Flow Cell
  • Pipeta y puntas P1000
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
CONSEJO

Priming and loading a flow cell

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

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.

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.

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.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

MEDIDA OPCIONAL

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.

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

Flow Cell Loading Diagrams Step 2

IMPORTANTE

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.

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.

Flow Cell Loading Diagrams Step 03 V5

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.

Flow Cell Loading Diagrams Step 04 V5

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

IMPORTANTE

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.

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.

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.

Flow Cell Loading Diagrams Step 5

Flow Cell Loading Diagrams Step 06 V5

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

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.

Flow Cell Loading Diagrams Step 07 V5

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.

Flow Cell Loading Diagrams Step 8

Flow Cell Loading Diagrams Step 9

11. Data acquisition and basecalling

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.

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 MinION Mk1B/Mk1D device

Follow the instructions in the MinION Mk1B user manual or the MinION Mk1D 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 GridION device

Follow the instructions in the GridION user manual.

5. 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.

6. 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.

IMPORTANTE

Settings in MinKNOW

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

12. Downstream analysis

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

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.

13. Flow cell reuse and returns

Material
  • Flow Cell Wash Kit (EXP-WSH004)

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°C to +8°C.

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

CONSEJO

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.

Alternatively, follow the returns procedure to send the flow cell back to Oxford Nanopore.

Instructions for returning flow cells can be found here.

IMPORTANTE

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.

14. Issues during DNA/RNA extraction and library preparation

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.

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.

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. SPRI cleanup
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.

15. Issues during the sequencing run

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.

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.

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.

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.

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. DNA gel2 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.

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)

image2022-3-25 10-43-25 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.

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.

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.

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.

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 link for more information on MinION temperature control.

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

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.

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.

Last updated: 3/10/2023

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