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Hi all -
I am planning an amplicon sequencing run on a NextSeq 550 that will require a custom read 1 sequencing primer. I am wondering if anyone has some experience and can comment on these two factors:
1) Tm - Illumina docs state the primer binding temp on the nextseq is 60 C, so what temp should be aimed for in designing the custom primer Tm if length is not an issue? I have run a successful custom primer run before with a primer Tm of 60 on the nextseq, but wondering if perhaps a few degrees above or below tends to give better results.
2) G/C clamp - In my last run, I had a G at the 3' end of the custom primer. The current design of this library places an A at the 3' end of the custom primer. Does the G/C clamp matter for custom primers on the NextSeq?
Thanks everyone,
Jordan
I am planning an amplicon sequencing run on a NextSeq 550 that will require a custom read 1 sequencing primer. I am wondering if anyone has some experience and can comment on these two factors:
1) Tm - Illumina docs state the primer binding temp on the nextseq is 60 C, so what temp should be aimed for in designing the custom primer Tm if length is not an issue? I have run a successful custom primer run before with a primer Tm of 60 on the nextseq, but wondering if perhaps a few degrees above or below tends to give better results.
2) G/C clamp - In my last run, I had a G at the 3' end of the custom primer. The current design of this library places an A at the 3' end of the custom primer. Does the G/C clamp matter for custom primers on the NextSeq?
Thanks everyone,
Jordan