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PCR Barcoding Expansion Pack 1-96 features
This kit is recommended for users who:
- want to optimise their sequencing experiment for throughput
- require control over read length
- would like to utilise upstream processes such as size selection or whole genome amplification
- want to multiplex up to 96 different samples
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Introduction to the PCR Barcoding Expansion Pack 1-96
This protocol describes how to carry out PCR barcoding using the Ligation Sequencing Kit 1D (SQK-LSK110) and the PCR Barcoding Expansion Pack 1-96 (EXP-PBC096). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity
The quality checks performed during the protocol are essential in ensuring experimental success
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the software for acquiring and analysing your data
- Check your flow cell to ensure it has enough pores for a good sequencing runLibrary preparation
You will need to:
- Prepare the DNA ends for adapter attachment
- Attach barcoding adapters supplied in the kit to the DNA ends
- Amplify each barcoded sample by PCR, then pool the samples together
- Repair the DNA, and prepare the DNA ends for adapter attachment
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cellSequencing 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
- Start the EPI2ME software and select the barcoding workflow -
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