# problems finalising sff.multiple analyses
**URL:** https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399
**Category:** Commands in mothur
**Created:** [June 19, 2013, 6:37am UTC](https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399 "2013-06-19T06:37:36Z")
**Posts on this page:** 4
**Page:** 1
### Author:  [@plaffy](https://forum.mothur.org/u/plaffy)
#### Post date: [June 19, 2013, 6:37am UTC](https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399/1 "2013-06-19T06:37:36Z")
Hi,
i was wondering if anyone else has come up with the same issue as i have encountered today
i am running a sff.multiple command on a project containing 20 different sequencing reactions (i have already completed this analysis on two alternate species with no issues, although sff files were produced from a separate sequencing run).
however, for the third time now, my analysis halts once the final shhh.flows, trim.seqs and summary.seqs commands have run from within sff.multiple. the last commandline output is as follows
\_Running command: trim.seqs(fasta=png10c3.shhh.fasta, name=png10c3.shhh.names, oligos=pngbacteria3.txt, allfiles=t, flip=f, keepforward=f, pdiffs=2, bdiffs=0, ldiffs=0, sdiffs=0, tdiffs=2, maxambig=-1, minlength=200, maxlength=0, processors=1)
Using 1 processors.
1000
1401
Group count:
png10c3 6967
Total of all groups is 6967
Output File Names:
png10c3.shhh.trim.fasta
png10c3.shhh.scrap.fasta
png10c3.shhh.trim.names
png10c3.shhh.scrap.names
png10c3.shhh.png10c3.fasta
png10c3.shhh.png10c3.names
png10c3.shhh.groups
png10c3.shhh.png10c3.groups
Running command: summary.seqs(fasta=png10c3.shhh.trim.fasta, processors=1, name=png10c3.shhh.trim.names)
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 200 200 0 3 1
2.5%-tile: 1 205 205 0 4 175
25%-tile: 1 224 224 0 5 1742
Median: 1 226 226 0 5 3484
75%-tile: 1 226 226 0 5 5226
97.5%-tile: 1 236 236 0 6 6793
Maximum: 1 249 249 0 7 6967
Mean: 1 223.711 223.711 0 4.91388
# of unique seqs: 1393
total # of seqs: 6967
Output File Names:
png10c3.shhh.trim.summary
/\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*/
plaffy@plaffy-Precision-T7600:~/projects/PNGproject/originaldata/work/bacteria/species3$\_
as you can see, the process jumps straight out of mothur and does not provide me with any error information so i am not sure why this error keeps occuring.
i thought this could be due to the original input file format or the.oligos file format, but i have checked these and cannot find the issue
i have also performed the analysis on the final sff file, and i have been able to complete these steps without issue
i have noticed there was a similar sounding post a week or so ago, but i cant seem to figure out what the issue is from that post
[http://mothur.ltcmp.net/t/shhh-flows-error-for-one-file/1296/6](http://mothur.ltcmp.net/t/shhh-flows-error-for-one-file/1296/6)
any help you can provide on solving this problem would be greatly appreciated
thanks
Patrick Laffy
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### Author:  [@westcott](https://forum.mothur.org/u/westcott)
#### Post date: [June 19, 2013, 11:09am UTC](https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399/2 "2013-06-19T11:09:01Z")
Could you post the contents of the file and oligos files, and your sff.multiple command?
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### Author:  [@plaffy](https://forum.mothur.org/u/plaffy)
#### Post date: [June 19, 2013, 10:21pm UTC](https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399/3 "2013-06-19T22:21:53Z")
the sff.multiple command is as follows
sff.multiple(file=pngbacteriashhh.txt, maxhomop=8, pdiffs=2, signal=0.60, noise=0.65, minflows=360, maxflows=360, order=A, processors=12, allfiles=T, minlength=200)
the pngbacteriashhh.txt file is as follows
png02b3.sff pngbacteria3.txt
png02b3.sff pngbacteria3.txt
png03b3.sff pngbacteria3.txt
png04b3.sff pngbacteria3.txt
png05b3.sff pngbacteria3.txt
png06b3.sff pngbacteria3.txt
png07b3.sff pngbacteria3.txt
png08b3.sff pngbacteria3.txt
png09b3.sff pngbacteria3.txt
png10b3.sff pngbacteria3.txt
png01c3.sff pngbacteria3.txt
png02c3.sff pngbacteria3.txt
png03c3.sff pngbacteria3.txt
png04c3.sff pngbacteria3.txt
png05c3.sff pngbacteria3.txt
png06c3.sff pngbacteria3.txt
png07c3.sff pngbacteria3.txt
png08c3.sff pngbacteria4.txt
png09c3.sff pngbacteria3.txt
png10c3.sff pngbacteria3.txt
there are two oligos files(pngbacteria3.txt and pngbacteria4.txt), as the data comes from two different sequencing runs and there was some overlap with the barcodes used:
pngbacteria3.txt was as follows
forward gagtttgatcntggctcag
barcode AACGTGGA png01b3
barcode AACGTGGC png02b3
barcode AACGTGGT png03b3
barcode AACGTGTA png04b3
barcode AACGCTTG png05b3
barcode AACGTGTC png06b3
barcode AACGGAAG png07b3
barcode GTATGTTC png08b3
barcode AACGGACA png09b3
barcode AACGGACC png10b3
barcode AACGGACG png01c3
barcode GTATTCAC png02c3
barcode AACGGAGA png03c3
barcode AACGTTAA png04c3
barcode AACGGAGG png05c3
barcode AACGGAGT png06c3
barcode AACGGATA png07c3
barcode AACGTTAC png09c3
barcode AACGTTAG png10c3
finally, pngbacteria4.txt was as follows
forward gagtttgatcntggctcag
barcode AACGTGGA png08c3
hopefully you can make something of this issue
thanks
Patrick
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### Author:  [@plaffy](https://forum.mothur.org/u/plaffy)
#### Post date: [June 20, 2013, 6:16am UTC](https://forum.mothur.org/t/problems-finalising-sff-multiple-analyses/1399/4 "2013-06-20T06:16:51Z")
apoligies for wasting your time,
i have found the problem, if you look in the file pngbacteriashhh.txt we have two lines pertaining to png02b3.sff pngbacteria3.txt and when this duplicate is removed the analysis works as it should
thanks for your quick response anyway
patrick