How 2-channel sequencing chemistry (Next-Seq) distinguish "G" and 'no signal'

Question

[CY]

We found some samples enriched with "G" and we think this may caused by 2-channel sequencing chemistry can't distinguishing "G" and 'no signal'.

However, we have not have chance to sequence lot of sample using Next-Seq. Is 'no signal' or 'N' never appears in Next-Seq output? This seems to be a really serious flaw to me. Does it make sense?

[GenoMax]

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Answer 1

[Devon Ryan]

You will commonly see an enrichment of GGGGGGGG sequences on a NextSeq for the reason you mentioned. As an example, when you demultiplex a run you will typically find GGGGGGGG to be the "unplaced barcode" with the highest count. This is, of course, actually NNNN. Yes, this is a known limitation of NextSeq machines. For that reason I wouldn't use them for things like BS-seq, where an individual G can have a lot of meaning.