What was your extracted [DNA]? triplicate PCRs or just one reaction per sample? Have you tried reextracting that sample or at least reamplifying the extract you have? I haven't worked on fish, but I'd expect an animal that has just mycoplasma in their gut to be fairly sick looking. I'd be surprised if the issue is in the sequencer when you're getting millions of reads (regardless if they were multiplexed with samples that were pure mycoplasma, you shouldn't get that much index jumping). I'd focus on wet lab contamination.
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Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct. -
Triplicate pcr's - a lot of studies pubkished recently on fish gut microbiome are finding mycoplasma, so I'm not sure if it's contamination. Plus we tested samples of the reagents as neg controls too and no myco. We did find a great diversity of otus too not just pure myco.
If 24 samples on one miseq flow cell, should produce over 500k reads per sample no? Considering illumina expects 100k per sample if 96 samples are run, as per their protocol?Comment
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