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Old 08-28-2017, 09:57 AM   #9
Location: Storrs, CT

Join Date: Sep 2012
Posts: 13
Default Still a little confused

I'm a bit confused about how to accomplish the dual-indexing using the same index on each end. My workflow ligates an adapter, and then uses indexing primers in PCR to get flow-cell adapters and indices onto the molecules. When I'm single indexing, this involves a "universal forward primer" and a unique reverse primer with the index. I can't just use the reverse primer without the universal forward primer, can I? I won't get indices on both ends, will I?

This seems awfully elementary, I know, but it's hard to keep track of exactly what protocol people are talking about doing. I 100% understand using two unique index primers for your library, out of, say, Illumina's TruSeq dual-indexing kit. But I don't understand how people are suggesting using the same index on both ends. You've got to be using custom oligos, right, with a forward and reverse primer that contain the same index region?

Are some sequencing cores now stocking indexing primers like this that customers can purchase? Because I sure as heck cannot afford 96 pairs of custom Illumina primers.
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