Applying pairwise.seqs for ITS1-ITS2

Commands in mothur
1

mothur > summary.seqs(count=stability.trim.contigs.good.count_table)

output:

Using stability.trim.contigs.good.unique.fasta as input file for the fasta parameter.

Using 20 processors.

            Start   End     NBases  Ambigs  Polymer NumSeqs

Minimum: 1 50 50 0 3 1
2.5%-tile: 1 167 167 0 4 327623
25%-tile: 1 248 248 0 4 3276221
Median: 1 441 441 0 5 6552441
75%-tile: 1 463 463 0 6 9828661
97.5%-tile: 1 467 467 0 8 12777259
Maximum: 1 500 500 0 8 13104880
Mean: 1 349 349 0 5

of unique seqs: 5940853

total # of seqs: 13104880

It took 63 secs to summarize 13104880 sequences.

Output File Names:
stability.trim.contigs.good.unique.summary

aligning the sequences you are analyzing

instructed by Pat Schloss

mothur > pairwise.seqs(fasta=stability.trim.contigs.good.unique.fasta)

As I ran this command, MOTHUR stays on for like 5 minutes then aboerting. Any idea why? How to rectify this?

2

Hi - it’s probably because you have close to 6 million sequences and it is using up all of the RAM on your computer.

Pat

1 Like
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I see! Is there any way to make it not happen? I was wondering if I’m running pairways.seq on the correct finger. That’s what I got after make.contigs, screen.seq and unique.seq. Would you suggest running pre.cluster and chimera.vsearch before running pairways.seq? I saw on the forum people did not use pairways.seq at all for their ITS reads. The ly just went on with pre.cluster and chinera.vsearch after unique.seq and then went in with cluster.otu with unite dataset

4

@westcott do you have any idea regarding my issue as well?

5

This is usually a data quality issue, which is likely to be more pronounced with ITS data. When the paired reads do not fully overlap you do not get adequate desnoising. For example, you have contigs that are 500 nt long which means there is no overlap between them. Those are almost certainly garbage. ITS analysis is fraught with a lot of problems that make me uneasy making many suggestions with paired illumina data.

Sorry!
Pat

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Hi Pat, thank you for understanding my situation. I’m still running the pre.cluster and then chimera.vsearch to move on to see what it gives me downstream. Hoping for the best :crossed_fingers:t3:

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