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?
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?