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  • Choosing the best hexamer sequences for custom adapters?

    I have two sets of adapters that I am using for my library preparations. Both are custom adapters.

    Universal Y-shaped Adapter 1:
    ACACTCTTTCCCTACACGACGCTCTTCCGATC*T
    Adapter 1 barcode:
    /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCXXXXXXTCTCGTATGCCGTCTTCTGCTTG
    IDT recommended barcodes:
    TATGCG
    AACACC
    AACCAG
    AACGGA
    AAGAGG
    AAGCCT
    AAGGTC
    AATCGC
    ACAACG
    ACCGAT

    I then PCR amplify these barcoded libraries, adding an extra barcode to the amplicon. I use the following primers:

    PCR primer 1:
    CAAGCAGAAGACGGCATACGAGA*T
    PCR primer adapters:
    AATGATACGGCGACCACCGAGATCTACACXXXXXXACACTCTTTCCCTACACG
    IDT recommended barcodes:
    ACCTCA
    ACGCAA
    ACGTGT
    ACTCTG
    AGAAGC
    AGCATG
    AGCTAC
    AGGACA
    AGTGAG
    ATGTCC

    My question is, does anyone have any experience in choosing the hexamer barcodes, for use with Next-Seq?

    I emailed IDT, and they recommended specific hexamer sequences (shown above). I simply want to know why these sequences were chosen, they have not been able to explain why. The only thing I can see, is the correct balance of AT to CG in the hexamer? Are there other considerations?

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