Hi QIIME2 folks! Happy holiday week!
I am troubleshooting some interesting-looking reverse reads. Our lab tried out some new primers (515f_Mod, 806r_Mod), which are modified from the EMP primers to include more degeneracy to capture more diversity (see Hu et al. 2024). These are in-house sequenced 2x150 Illumina Miseq reads. Our forward reads look relatively normal, with a consistent drop in quality, but the quality of the reverse reads goes down, back up, and down again, which is quite unusual. Since the forward read quality looks normal I don’t think it’s a bioinformatics issue, and the reads per sample for forward and reverse reads look good (see attached demux.qzv).
demux.qzv (332.6 KB)
A colleague mentioned this could just be a sequencer issue, which to me seems most likely. Has anyone used these primers before, and if so, did you have reverse read quality like this? Or has anyone seen reverse quality like this (with forward reads looking normal) and found out it was a sequencer issue? We’ve contacted Illumina to see what they say, but I’m curious if others have seen this (and I didn’t see a previous post on this topic, forgive me if there has already been one).
Any insight is helpful!
Heather Deel
I’ve never seen one that produced reverse reads like this before!