shhh.trim.fasta is blank

Hello Mothur forum,

I have 454 pyrosequencing data. I have used sff procedure, below. My shhh.trim.fasta file is blank, minlength=400 (trim.seqs). How do I get longer sequences?
Thanks
Kirsi


mothur v.1.25.0 Last updated: 5/1/2012

mothur > sffinfo(sff=t1.sff, flow=T)
Output File Names:
t1.fasta
t1.qual
t1.flow

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 39 39 0 2 1
2.5%-tile: 1 62 62 0 3 349
25%-tile: 1 230 230 0 4 3486
Median: 1 538 538 0 5 6971
75%-tile: 1 559 559 0 5 10456
97.5%-tile: 1 581 581 0 6 13593
Maximum: 1 700 700 4 9 13941
Mean: 1 419.908 419.908 0.0105444 4.62564

of Seqs: 13941

Output File Name:
t1.fasta.summary

mothur > trim.flows(flow=t1.flow, oligos=t1.oligos, pdiffs=2, bdiffs=1, processors=2)

Output File Names:
t1.trim.flow
t1.scrap.flow
t1.Spring.flow
t1.flow.files

mothur >
shhh.flows(file=t1.flow.files, processors=2)
Output File Names:
t1.Spring.shhh.qual
t1.Spring.shhh.fasta
t1.Spring.shhh.names
t1.Spring.shhh.counts
t1.Spring.shhh.groups
t1.shhh.fasta
t1.shhh.names

mothur > trim.seqs(fasta=t1.shhh.fasta, name=t1.shhh.names, oligos=t1.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=400, flip=T, processors=2)
Output File Names:
t1.shhh.trim.fasta
t1.shhh.scrap.fasta
t1.shhh.trim.names
t1.shhh.scrap.names
t1.shhh.groups

mothur > trim.seqs(fasta=t1.shhh.fasta, name=t1.shhh.names, oligos=t1.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=400, flip=T, processors=2)

1000
1000
2000
2072
2000
2065
Appending files from process 21513

Output File Names:
t1.shhh.trim.fasta
t1.shhh.scrap.fasta
t1.shhh.trim.names
t1.shhh.scrap.names
t1.shhh.groups

mothur > summary.seqs(fasta=t1.shhh.trim.fasta, name=t1.shhh.trim.names)

[ERROR]: t1.shhh.trim.names is blank. Please correct.
[ERROR]: t1.shhh.trim.fasta is blank. Please correct.
Error in reading your fastafile, at position -1. Blank name.
Segmentation fault

After running trim.flows and shhh.flows, your sequences will be ~250 bp, none of them will be longer than 400. Roche’s numbers are deceiving - as we showed in our recent PLoS ONE paper, sequence quality dies off after 200-250 bp.

Pat