While checking some Mothur output, I’ve come across two places where I don’t understand what Mothur is doing. The first is in make.contigs, the second is in pcr.seqs (that question is in a separate post).
When I look at a random sample of raw reads, I see the exact primer match at the start of R1 (forward) and R2 (reverse primer), as expected. I also see an exact match for the reverse complement of the reverse primer in R1, and an imperfect match for the reverse complement of the forward primer in R2. The imperfect forward primer has one mismatch.
I see that the imperfect forward primer appears in the output of make.contigs. So, why does Mothur, when merging, “capture” the imperfect forward primer when a perfect one exists?
I found this to be the case for about 50% of my random sample of reads. It leads me to believe I don’t quite understand how make.contigs works. Here are the parameters I run:
make.contigs(file=Undetermined.paired.files, processors=40, format=illumina1.8+, oligos=amr.oligos, bdiffs=0, pdiffs=0, checkorient=t, insert=25, trimoverlap=f, allfiles=0)