Hi. We are using a transposon sequencing (Tn-seq) protocol that uses a custom sequencing primer to sequence through the transposon into the genomic DNA of interest. The lack of diversity in the transposon sequence is causing problems on HiSeq runs. We are considering changing the protocol to incorporate something like the Illumina universal adapter during library prep and I have some questions about how to resolve the low diversity issue (as we would still like to sequence through the transposon). I know that staggered primers can be used to introduce diversity. My question is whether using a single primer with a 'variable' region (6-8 N's introduced during oligo synthesis) would also work? In this case, one would sequence through the variable region, then into the low diversity region, then into the high diversity region of interest. Use of a single primer would be more cost effective than staggered primers. Does anyone have any experience or thoughts on the matter?
Thanks.
Thanks.
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