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Old 06-04-2018, 11:34 AM   #4
Location: Canada

Join Date: Jun 2013
Posts: 56

Hi again Sean,

Sorry for making that guess/assumption. I forgot that eukaryotes have 16S RNA as well.

So it does appear that you have very low base diversity, you have rapidly declining q scores that appear to coincide with increasing %G at base 120 of read 1, and most of your clusters passing filter come from lower densities (an indicator of overclustering). I am still leaning toward your troubles being from low-diversity and moderate overclustering but it could also be from bad reagents or something else.

This video goes over a lot of the same things as the document I added earlier:

I have lost one amplicon run to an expired kit which gave us a bunch of junk G nucleotides and rapidly declining q-scores. A re-run with the same library with new MiSeq reagents gave us excellent data. That being said, I have seen some colleagues who do not stagger/offset and/or mix in other loci get worse %PF and Qscores than I do with the same sequencing provider.

I have included 3 different offset/staggered amplicons in the 5 MiSeq amplicon runs I have done and they have all worked well with ~10-15% PhiX(aside from a 6th run that had the bad reagents). Perhaps someone with more experience interpreting SAV results will be able to tell you which problem your results are indicative of.

Some screen shots from FastQC would likely help with diagnosis as well.
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