We’ve done a couple of runs using the MiSeq 2x300 bp using the 27F and 519R primer set. Our first couple of runs seemed OK, but our latest one is dodgy as. I believe getting this region to work well really depends if you get a good sequencing run. I would highly recommend NOT sequencing this region and use the V4 region (and have a consensus reads as an additional quality control as in the paper - listen to Pat! :lol: ).
Given our dodgy run, I was trying to delve deeper in to the make.contigs command. I used fastQC to look at the quality of the calls in the contigs (make.fastq then fastQC), although I just came across a post here about the quality calls being calculated by PandaSeq so I’m not sure what the quality value mean now (i attached the pictures). It is a little bit unclear to me whether make.contigs does some trimming before alignment of contigs given the “trim” is in the resulting file name. So;
How does the trimming work in make.contigs?
How should we deal with the quality values here? I compared them to a V4 run we did (note: the contigs have not been screened, so they include the dodgey ones).
V123 - R1 (300 bases)
V123 - R2 (300 bases)
V123 - contig (600 bases)
V4 - R1 (250 bases)
V4 - R2 (250 bases)
V4 - contig (500 bases)
V4 - contig (cut to 275 bases)