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Old 04-23-2020, 06:33 AM   #7
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Location: Boston, MA, USA

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Originally Posted by ATϟGC View Post
If these are amplicon libraries and you want to minimize the amount of PhiX you can add "stagger" or "offset" nucleotides between the illumina sequencing primer region (like the nextera or truseq tail) and your locus-specific primer in order to create diversity of bases. These stagger nucleotides can also be added to restriction-digests adapters to increase base diversity.

I always add staggers to my amplicon primers and sequence multiple amplicons per run to increase diversity but I still always add 5-12% Phix just to be sure.
Thanks, ATϟGC, that's a good suggestion.
Looking back, the adapter-primers that I had used for my older runs when I didn't have this issue, did have some degenerate bases in between for different purposes and I think that was key in preventing this issue.

Still adding a minimal portion of PhiX is a good suggestion, too.
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