Align sequence with no primers

Theory behind mothur
1

Dear all,
May I perform the align.seqs using samples without primers+adapters (we’ve sequenced in BGI facilities and they shared the CleanData without primers) and Silva reference excluding primer sequence?
I’m following the steps according to SOP.
I’ve done it and the summary seqs return like this:

mothur > align.seqs(fasta=luan.trim.contigs.good.unique.fasta, reference=silva.nr_v138_1.pcr.align)

Using 64 processors.

Reading in the silva.nr_v138_1.pcr.align template sequences… DONE.
It took 19 to read 146601 sequences.

Aligning sequences from luan.trim.contigs.good.unique.fasta …
It took 36 secs to align 117429 sequences.

[WARNING]: 3 of your sequences generated alignments that eliminated too many bases, a list is provided in luan.trim.contigs.good.unique.flip.accnos.
[NOTE]: 1 of your sequences were reversed to produce a better alignment.

It took 37 seconds to align 117429 sequences.

Output File Names:
luan.trim.contigs.good.unique.align
luan.trim.contigs.good.unique.align_report
luan.trim.contigs.good.unique.flip.accnos

mothur > summary.seqs(fasta=current, count=luan.trim.contigs.good.count_table)
Using luan.trim.contigs.good.unique.align as input file for the fasta parameter.

Using 64 processors.

	Start	End	NBases	Ambigs	Polymer	NumSeqs

Minimum: 1 18 7 0 3 1
2.5%-tile: 1 9583 253 0 4 26699
25%-tile: 1 9583 253 0 5 266984
Median: 1 9583 253 0 5 533967
75%-tile: 1 9583 253 0 6 800950
97.5%-tile: 1 9583 253 0 6 1041235
Maximum: 8716 9583 270 0 8 1067933
Mean: 1 9582 252 0 5

of unique seqs: 117429

total # of seqs: 1067933

2

Hi Allan,

That looks right. You’ll want to run screen.seqs next to remove the short sequences.

Pat

3

Hi Pat,
Thanks for replying.
Then I can’t proceed with align.seqs command without the primers using this ‘clean data’? Should I run align seqs with the primers and then remove them with screen seqs?

Thanks

4

Can you remove the primers and barcodes in make.contigs? You’ll definitely want to know which sample each sequence belongs to.

Pat

5

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