align.seqs reverse complement

Commands in mothur
1

hi,
I ran an alignment on my sequences and when I look in the flip.accnos file, it appears that the alignment used all of my sequences in the reverse complement. Is this common and should I be concerned that my alignment may not be accurate? (Using v.13.0) Here is the summary of my sequences before the alignment:

mothur > summary.seqs(fasta=dennisLabeledFinal.pick.trim.unique.fasta)

Start End NBases Ambigs Polymer
Minimum: 1 350 350 0 3
2.5%-tile: 1 358 358 0 4
25%-tile: 1 412 412 0 5
Median: 1 468 468 0 5
75%-tile: 1 509 509 0 5
97.5%-tile: 1 526 526 0 6
Maximum: 1 562 562 0 8

of Seqs: 52131

and summary after alignment : mothur > align.seqs(candidate=dennisLabeledFinal.pick.trim.unique.fasta, template=silva.bacteria.fasta, flip=T)

Start End NBases Ambigs Polymer
Minimum: 1044 1062 9 0 3
2.5%-tile: 1044 6434 339 0 4
25%-tile: 1044 8419 393 0 5
Median: 1044 10259 449 0 5
75%-tile: 1044 13858 489 0 5
97.5%-tile: 1044 13862 507 0 6
Maximum: 40159 43116 543 0 8

of Seqs: 52131

Thanks!

2

I suspect you sequenced off of the reverse primer. People are doing this commonly with the v13 and v35 regions. They’ll sequence off of the reverse primer and so the sequences need to be flipped. You can speed things up a bit by using the flip option in trim.seqs and dropping it from align.seqs