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Old 04-24-2020, 05:05 AM   #10
ATϟGC
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Location: Canada

Join Date: Jun 2013
Posts: 56
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I agree that would be best to discuss these issues with your sequencing provider.

If you do choose to use staggered bases I recommend making an alignment to check for base diversity in the first 12-20 base pairs of read1. This alignment should be made with respect to the Illumina sequencing primer. For my amplicon libraries, this means I anchor it on the left by the Nextera Read1 sequences. You then only need to consider the base diversity of your staggered and/or unstaggered (I use a mix of both in my round 1 PCR reactions) primers or adapters. I do this in microsoft excel so that I can calculate and optimize base diversity of all the amplicons that will be pooled in my run.

Adding stagger bases has the potential to introduce biases in your libraries due to secondary structures or other priming phenomena. If you use the same mix of staggers for all samples the bias should be the same in theory.

I have only sequenced amplicons on Miseq and Novaseq and 5-12% PhiX has been enough for me with those platforms so I cannot comment on Hiseq.
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