- Materials
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- 100-200 fmol first-round PCR product in 48 μl (with tailed primers, see full protocol for further details)
- PCR Barcoding Expansion 1-12 (EXP-PBC001)
- Ligation Sequencing Kit (SQK-LSK112)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Thermal cycler
- 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
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
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For this protocol, you will need 100-200 fmol first-round PCR product with tailed primers in 48 μl.
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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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NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.
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Ligation Sequencing Kit (SQK-LSK112) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) DNA CS DCS Yellow 1 35 Adapter Mix H AMX H Green 2 40 AMPure XP Beads AXP Amber 1 1,200 Ligation Buffer LNB Clear 1 200 Long Fragment Buffer LFB Orange 2 1,800 Short Fragment Buffer SFB Clear 2 1,800 Sequencing Buffer II SBII Red 1 500 Elution Buffer EB Black 1 1,200 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink sticker on label 1 400 Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200 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.
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PCR Barcoding Expansion 1-12 (EXP-PBC001) contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) PCR Barcode 1-12 BC1-12 Clear 12 20 Barcode Adapter BCA Blue stripe 1 260 -
Rapid barcode sequences
Below is a full list of the rapid barcode sequences used across the rapid-based barcoding kits, with the component acronym for the specific sequencing kit. The barcodes in our rapid-based kits are shipped at 10 µM.
Note: These are not the full sequences due to proprietory information.
In addition to the barcodes, there are also flanking sequences which add an extra level of context during analysis.
Rapid barcoding kits
Rapid Barcoding Kit 24 V14 (SQK-RBK114.24): RB01-24
Rapid Barcoding Kit 96 V14 (SQK-RBK114.96): RB01-96
Legacy: Rapid Barcoding Kit (SQK-RBK004): RB01-12
Rapid Barcoding Kit 96 (SQK-RBK110.96): RB01-96Barcode flanking sequence:
5' - GCTTGGGTGTTTAACC - barcode - GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTTCA - 3'
PCR barcoding kits
PCR-cDNA Barcoding Kit 24 V14 (SQK-PCB114.24): BP01-24
Legacy PCR-cDNA Barcoding Kit 24 (SQK-PCB111.24): BP01-24
Legacy: PCR-cDNA Barcoding Kit (SQK-PCB109): BP01-12
Legacy: PCR Barcoding Kit (SQK-PBK004): BP01-12Barcode flanking sequence:
Top strand: 5' - ATCGCCTACCGTGA - barcode - TTGCCTGTCGCTCTATCTTC - 3'
Bottom strand: 5' - ATCGCCTACCGTGA - barcode - TCTGTTGGTGCTGATATTGC - 3'The top and bottom barcode flanking sequences are different to avoid 5' and 3' end sequences annealing to each other and forming a loop.
16S barcoding kits
16S Barcoding Kit 24 V14 (SQK-16S114.24): 16S01-24
Legacy 16S Barcoding Kit 1-24 (SQK-16S024): 16S01-24
Legacy: 16S Barcoding Kit (SQK-RAB204): 16S01-12Barcode flanking sequence:
Forward 16S primer: 5' - ATCGCCTACCGTGAC - barcode - AGAGTTTGATCMTGGCTCAG - 3'
Reverse 16S primer: 5' - ATCGCCTACCGTGAC - barcode - CGGTTACCTTGTTACGACTT - 3'The 3' flanking sequence of the forward primer contains a wobble base (denoted by M; in the primer the base is either an A or a C) in a variable region of the 16S gene.
Rapid PCR barcoding kits
Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24): RLB01-24
Legacy: Rapid PCR Barcoding Kit (SQK-RPB004): RLB01-12ABarcode flanking sequence:
5' - ATCGCCTACCGTGAC - barcode - CGTTTTTCGTGCGCCGCTTC - 3'
PCR barcoding expansions
PCR Barcoding Expansion 1-12 (EXP-PBC001): BC01-12
PCR Barcoding Expansion 1-96 (EXP-PBC096): BC01-96Barcode flanking sequence:
5' - GGTGCTG - barcode - TTAACCT - 3'Component Sequence BP01 / BC01 / RB01 / 16S01 / RLB01 AAGAAAGTTGTCGGTGTCTTTGTG BP02 / BC02 / RB02 / 16S02 / RLB02 TCGATTCCGTTTGTAGTCGTCTGT BP03 / BC03 / RB03 / 16S03 / RLB03 GAGTCTTGTGTCCCAGTTACCAGG BP04 / BC04 / RB04 / 16S04 / RLB04 TTCGGATTCTATCGTGTTTCCCTA BP05 / BC05 / RB05 / 16S05 / RLB05 CTTGTCCAGGGTTTGTGTAACCTT BP06 / BC06 / RB06 / 16S06 / RLB06 TTCTCGCAAAGGCAGAAAGTAGTC BP07 / BC07 / RB07 / 16S07 / RLB07 GTGTTACCGTGGGAATGAATCCTT BP08 / BC08 / RB08 / 16S08 / RLB08 TTCAGGGAACAAACCAAGTTACGT BP09 / BC09 / RB09 / 16S09 / RLB09 AACTAGGCACAGCGAGTCTTGGTT BP10 / BC10 / RB10 / 16S10 / RLB10 AAGCGTTGAAACCTTTGTCCTCTC BP11 / BC11 / RB11 / 16S11 / RLB11 GTTTCATCTATCGGAGGGAATGGA BP12 / BC12 / RB12 / 16S12 CAGGTAGAAAGAAGCAGAATCGGA RLB12 (in current chemistry kits V14+ / RLB012A in legacy kits) GTTGAGTTACAAAGCACCGATCAG BP13 / BC13 / RB13 / 16S13 / RLB13 AGAACGACTTCCATACTCGTGTGA BP14 / BC14 / RB14 / 16S14 / RLB14 AACGAGTCTCTTGGGACCCATAGA BP15 / BC15 / RB15 / 16S15 / RLB15 AGGTCTACCTCGCTAACACCACTG BP16 / BC16 / RB16 / 16S16 / RLB16 CGTCAACTGACAGTGGTTCGTACT BP17 / BC17 / RB17 / 16S17 / RLB17 ACCCTCCAGGAAAGTACCTCTGAT BP18 / BC18 / RB18 / 16S18 / RLB18 CCAAACCCAACAACCTAGATAGGC BP19 / BC19 / RB19 / 16S19 / RLB19 GTTCCTCGTGCAGTGTCAAGAGAT BP20 / BC20 / RB20 / 16S20 / RLB20 TTGCGTCCTGTTACGAGAACTCAT BP21 / BC21 / RB21 / 16S21 / RLB21 GAGCCTCTCATTGTCCGTTCTCTA BP22 / BC22 / RB22 / 16S22 / RLB22 ACCACTGCCATGTATCAAAGTACG BP23 / BC23 / RB23 / 16S23 / RLB23 CTTACTACCCAGTGAACCTCCTCG BP24 / BC24 / RB24 / 16S24 / RLB24 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