mothur “#pcr.seqs(inputdir=./data3/, fasta=silva.full_v123.fasta, start=11895, end=25318, keepdots=F, processors=8)”
This results in predominantly alignments that start at 2 and end at 13423, with no. of aligned bases (NBases) to be predominantly 291. I rename the outcome of this step:
mv data3/silva.full_v123.pcr.fasta data3/silva.full_v123.v4.fasta
However, my align.seqs() step produces a similar predominant NBases value (291), which is of concern to me because I expect an aligned length typical of 16S V4 region (e.g., 259 or around this value).
mothur “#align.seqs(inputdir=./data3/, fasta=test.trim.contigs.good.unique.fasta, reference=silva.full_v123.v4.fasta, processors=8)”
What could be happening here? Thank you.
Primers I use are the following:
All sequences 5’-3’ orientation:
16S V4 Forward :: GTGCCAGCMGCCGCGGTAA
16S V4 Reverse :: GGACTACHVGGGTWTCTAAT