Choosing the best hexamer sequences for custom adapters? - SEQanswers

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anishdattani1

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Join Date: Jun 2017

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Choosing the best hexamer sequences for custom adapters?

06-26-2017, 10:27 AM

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?
Tags: barcodes, illumina, next-seq

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