pcr.seqs, align.seqs :: Why I observe length discrepancy?

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.

Additional information:

Primers I use are the following:
All sequences 5’-3’ orientation:

16S V4 Forward :: GTGCCAGCMGCCGCGGTAA
16S V4 Reverse :: GGACTACHVGGGTWTCTAAT

You probably still have your primers (and possibly the index sequences) attached to your sequences. You can look at some of the sequences coming out of test.trim.contigs.good.unique.fasta and see wehther those sequences are still attached. If they are, then you need to supply make.contigs with an oligos file.

Pat