- Materials
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- <100–200 fmol of each DNA sample to be barcoded in 45 µl
- OR <100–200 fmol first-round PCR product (with tailed primers) per sample
- PCR Barcoding Expansion 1-96 (EXP-PBC096)
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- Consumables
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- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- Freshly prepared 80% ethanol in nuclease-free water
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Microfuge
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Vortex mixer
- Thermal cycler
- Magnetic rack
- Multichannel pipette and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Eppendorf 5424 centrifuge (or equivalent)
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For gDNA input, <100–200 fmol of each DNA sample to be barcoded in 45 µl is required.
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For amplicon input, start from the "Barcoding PCR" step with <100–200 fmol first-round PCR product with tailed primers in the following volumes per sample:
- 48 μl for up to 12 barcodes
- 24 μl for 13 barcodes or more
Start from the "Barcoding PCR" step after the amplicons have undergone a first round of PCR to incorporate the following 5' tail sequences:
5’ TTTCTGTTGGTGCTGATATTGC-[ project-specific forward primer sequence ] 3’
5’ ACTTGCCTGTCGCTCTATCTTC-[ project-specific reverse primer sequence ] 3’The sequences above are required for the Barcoding PCR step to incorporate the Oxford Nanopore barcode sequences into your amplicon. Multiple first-round PCR products can be pooled together and we recommend all amplicon samples to receive the same barcode should be quantified and pooled in the desired ratios before the barcoding PCR step is performed.
After the first round of PCR to incorporate the 5' tail is complete, a purification step is required to remove any proteins, salts, dNTPs and primers. We recommend using AMPure XP Beads but other methods suitable for the amplicon size may also be used.
If you do not want to redesign your primers, the Barcode Adapter (BCA) from the PCR Barcoding Expansion 1-12 or 1-96 (EXP-PBC001 or EXP-PBC096) can be ligated onto the ends of the amplicons and the protocol started from the first end-prep step.
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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.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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PCR Barcoding Expansion Pack 1-96 (EXP-PBC096)
Name Acronym Cap colour No. of vials/plates Fill volume per well (µl) PCR Barcode Primer Mix plate BC01-96 White 1 plate 24 Barcode Adapter plate BCA Blue 1 plate 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.
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Capping and decapping the 96 well format
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Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format and reducing the concentration of EDTA.
Single-use tubes format with higher EDTA concentration:
Higher concentration of EDTA with a clear cap.Bottle format with reduced EDTA concentration:
Reduced concentration of EDTA with a blue cap.Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
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To maximise the use of the Native Barcoding Kits, the Native Barcode Auxiliary V14 (EXP-NBA114) and the Sequencing Auxiliary Vials V14 (EXP-AUX003) expansion packs are available.
These expansions provide extra library preparation and flow cell priming reagents to allow users to utilise any unused barcodes for those running in smaller subsets.
Both expansion packs used together will provide enough reagents for 12 reactions. For customers requiring extra EDTA to maximise the use of barcodes, we recommend using 0.25 M EDTA and adding 4 µl for library preps using the SQK-NBD114.24 kit.
Native Barcode Auxiliary V14 (EXP-NBA114) contents:
Note: This Product contains AMPure XP Reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Sequencing Auxiliary Vials V14 (EXP-AUX003) contents:
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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
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