# FLX+ SOP

**URL:** <https://forum.mothur.org/t/flx-sop/1626>\
**Category:** Commands in mothur\
**Created:** [December 24, 2013, 3:09am UTC](https://forum.mothur.org/t/flx-sop/1626 "2013-12-24T03:09:01Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![danieln](https://avatars.discourse-cdn.com/v4/letter/d/c6cbf5/32.png) [@danieln](https://forum.mothur.org/u/danieln)\
**Post date:** [December 24, 2013, 3:09am UTC](https://forum.mothur.org/t/flx-sop/1626/1 "2013-12-24T03:09:01Z")

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Hi,

I have a recent sff file generated through V1-4 FLX+ amplicon chemistry from my sequence provider.

Now if I fed it into the initial SOP commands:  
sffinfo(sff=sample.sff, flow=T)  
trim.flows(flow=sample.flow, oligos=sample.oligos, pdiffs=2, bdiffs=1, minflows=1050, maxflows=1050, order=B, processors=5)  
shhh.flows(file=sample.flow.files) \<— approximately 11-hours run (lookup file: GS FLX Titanium)  
trim.seqs(fasta=sample.shhh.fasta, name=sample.shhh.names, oligo=sample.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T)  
summary.seqs(fasta=sample.shhh.trim.fasta, name=sample.shhh.trim.names)

Plus if I try to proceed further into summary.seqs() here, I get an error prompt mentioning blank input files.

So this left me no choice but to find an alternative quality score approach:  
trim.seqs(fasta=sample.fasta, oligos=sample.oligos, qfile=sample.qual, maxambig=0, maxhomop=8, flip=T, bdiffs=1, pdiffs=2, qwindowaverage=35, qwindowsize=50) \<— “no namefile provided, fasta closely match with name=sample.shhh.trim.names”  
summary.seqs(fasta=sample.trim.fasta, name=sample.trim.names)  
unique.seqs(fasta=sample.trim.fasta, name=sample.trim.names)  
summary.seqs(fasta=sample.trim.unique.fasta, name=sample.trim.unique.names)  
align.seqs(fasta=sample.trim.unique.fasta, reference=silva.bacteria.fasta)  
summary.seqs(fasta=sample.trim.unique.align, name=sample.trim.unique.names)  
screen.seqs(fasta=sample.trim.unique.align, name=sample.shhh.trim.unique.names, group=sample.groups, end=27659, optimize=start, criteria=95)  
summary.seqs(fasta=sample.trim.unique.good.align, name=sample.trim.unique.good.names) \<— “blank input files, aborting…command not complete”

I just can’t find any solutions here onward (literally buried in a pile of technical problems I know I feel completely unaware of).

Anybody around for a desperate help, please?? 😢😢😢

Daniel

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**Author:** ![Patg13](https://avatars.discourse-cdn.com/v4/letter/p/d2c977/32.png) [@Patg13](https://forum.mothur.org/u/Patg13)\
**Post date:** [January 3, 2014, 7:23pm UTC](https://forum.mothur.org/t/flx-sop/1626/2 "2014-01-03T19:23:50Z")

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You must use the order=b parameter in the shhh.flows command or the trim.seqs command will dump all your sequences (blank input file error)  
I had the same problem and it work now !

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**Author:** ![danieln](https://avatars.discourse-cdn.com/v4/letter/d/c6cbf5/32.png) [@danieln](https://forum.mothur.org/u/danieln)\
**Post date:** [January 5, 2014, 9:28am UTC](https://forum.mothur.org/t/flx-sop/1626/3 "2014-01-05T09:28:44Z")

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Thanks, appreciate your help! Worked out all well!