My lab does a lot of MiSeq sequencing of low-diversity amplicon libraries and we've been using a standard 10% PhiX spike-in with reasonable results. I just heard about the idea of adding a few N's immediately downstream of the sequencing primer landing site, such that the first few bases are very high-diversity and the software can easily discern clusters before the low-diversity hits. We're considering switching our low-diversity assays to this N-based system, but I have a question before I order oligos.
Does this solution only require that the first few bases of read 1 have N's (i.e. high diversity) or do the first few bases of every read need this high diversity sequence? I can't imagine Illumina would store cluster positions from the first cycles of read 1 rather than having the software re-call clusters for each read, but I just wanted to check to see if anyone has experience with this type of low-diversity workaround before using a flow cell to test out my hypothesis.
Does this solution only require that the first few bases of read 1 have N's (i.e. high diversity) or do the first few bases of every read need this high diversity sequence? I can't imagine Illumina would store cluster positions from the first cycles of read 1 rather than having the software re-call clusters for each read, but I just wanted to check to see if anyone has experience with this type of low-diversity workaround before using a flow cell to test out my hypothesis.
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