# trim.flows on FLX+ or Junior 3.0 data
**URL:** https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853
**Category:** Commands in mothur
**Created:** [May 16, 2014, 5:32pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853 "2014-05-16T17:32:17Z")
**Posts on this page:** 9
**Page:** 1
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 16, 2014, 5:32pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/1 "2014-05-16T17:32:17Z")
I’m trying to trim 100,000 reads and I don’t seem to get any output. I created the oligos file by hand and I’m pretty sure I have the primers and barcodes right. Grep for them at the begining of the reads checks out ok. So does visual inspection via VIM and Less. Inspection was done via copy and paste from the oligos file so typos not a factor. Is the older Titanium lookup file OK on FLX+ / Junior 3.0???
> -rw-rw-r-- 1 genome genome 4 May 16 13:07 IJM7N3301.trim.flow
> -rw-rw-r-- 1 genome genome 835M May 16 13:08 IJM7N3301.scrap.flow
> -rw-rw-r-- 1 genome genome 0 May 16 13:08 IJM7N3301.flow.files
My oligo file #1 (w/ primer)
> forward TCAATTCNTTT
> barcode AGACCTCCCG long-13.8k-
> barcode TCGCGGCCCG long-12.9k-
> barcode TGAAGCCCGT long-9.56k-
> barcode AGACACCCGT long-9.37k-
> barcode ATACCACCCG long-8.03k-
> barcode TCACACCCGT long-7.08k-
> barcode TTCTCAACCC long-6.96k-
> barcode ACGCGCCCGT long-6.92k-
> barcode ACTCACCCGT long-6.82k-
> barcode TGGTGAACCC long-6.73k-
> barcode AACCTGGCCC long-6.61k-
> barcode TCTCCGTCCC long-6.57k-
My oligo file #2 (w/o primer)
$ cat IJM7N3301.oligos
> barcode AGACCTCCCG long-13.8k-
> barcode TCGCGGCCCG long-12.9k-
> barcode TGAAGCCCGT long-9.56k-
> barcode AGACACCCGT long-9.37k-
> barcode ATACCACCCG long-8.03k-
> barcode TCACACCCGT long-7.08k-
> barcode TTCTCAACCC long-6.96k-
> barcode ACGCGCCCGT long-6.92k-
> barcode ACTCACCCGT long-6.82k-
> barcode TGGTGAACCC long-6.73k-
> barcode AACCTGGCCC long-6.61k-
> barcode TCTCCGTCCC long-6.57k-
cat IJM7N3301.trim.flow
> 450
cat mothur.1400260070.logfile
> mothur \> trim.flows(flow=IJM7N3301.flow, oligos=IJM7N3301.oligos, pdiffs=1, bdiffs=0, processors=8)
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> Using 8 processors.
> 10000
> 13060
> 13060
> 13059
> 13060
> 13059
> 13060
> 13060
> 13060
> Appending files from process 6802
> Appending files from process 6803
> Appending files from process 6804
> Appending files from process 6805
> Appending files from process 6806
> Appending files from process 6807
> Appending files from process 6808
> Output File Names:
> IJM7N3301.trim.flow
> IJM7N3301.scrap.flow
> IJM7N3301.flow.files
> mothur \> quit()
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [May 16, 2014, 8:26pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/2 "2014-05-16T20:26:28Z")
In trim.flows you need to set flow=B. You might want to check this out too…
---
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 19, 2014, 4:30pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/3 "2014-05-19T16:30:47Z")
I’ll assume flow=B was a typo because that’s a true / false flag. Order B seems to work though. Thanks pat!
../../mothur/mothur "#sffinfo(sff=IJM7N3301.sff, flow=T)" ../../mothur/mothur "#summary.seqs(fasta=IJM7N3301.fasta)" ../../mothur/mothur "#trim.flows(flow=IJM7N3301.flow, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, processors=8, [b][/b])"
---
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 20, 2014, 6:29pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/4 "2014-05-20T18:29:32Z")
Going over the video now but all my sequences are going to scrap. My commands are below:
> …/…/mothur/mothur “#sffinfo(sff=IJM7N3301.sff, flow=T)”
> …/…/mothur/mothur “#summary.seqs(fasta=IJM7N3301.fasta)”
> …/…/mothur/mothur “#trim.flows(flow=IJM7N3301.flow, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, processors=8, order=B, minflows=1050, maxflows=1050)”
> …/…/mothur/mothur “#shhh.flows(file=IJM7N3301.flow.files, processors=8, lookup=…/…/mothur/LookUp\_Titanium.pat)”
> …/…/mothur/mothur “#trim.seqs(fasta=IJM7N3301.shhh.fasta, name=IJM7N3301.shhh.names, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T, processors=8)”
> …/mothur/mothur “#summary.seqs(fasta=IJM7N3301.shhh.trim.fasta, name=IJM7N3301.shhh.trim.names)”
> …/mothur/mothur “#summary.seqs()”
probably not productive but I’ve tried this as well
> …/…/mothur/mothur “#trim.seqs(fasta=IJM7N3301.shhh.fasta, name=IJM7N3301.shhh.names, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=1050, flip=T, processors=8)”
File size rundown:
> # ===================================================================== OUTPUT FROM COMMAND: sffinfo(sff=IJM7N3301.sff, flow=T)
>
> # 95M IJM7N3301.fasta 280M IJM7N3301.qual 834M IJM7N3301.flow
>
> # OUTPUT FROM COMMAND: summary.seqs(fasta=IJM7N3301.fasta)
>
> # 3.1M IJM7N3301.summary
>
> # OUTPUT FROM COMMAND: trim.flows(flow=IJM7N3301.flow, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, processors=8, order=B, minflows=1050, maxflows=1050)
>
> # 522M IJM7N3301.trim.flow 6.6M IJM7N3301.scrap.flow 72M IJM7N3301.long-13.8k-.flow 67M IJM7N3301.long-12.9k-.flow 50M IJM7N3301.long-9.56k-.flow 49M IJM7N3301.long-9.37k-.flow 42M IJM7N3301.long-8.03k-.flow 37M IJM7N3301.long-7.08k-.flow 36M IJM7N3301.long-6.96k-.flow 36M IJM7N3301.long-6.92k-.flow 35M IJM7N3301.long-6.82k-.flow 35M IJM7N3301.long-6.73k-.flow 35M IJM7N3301.long-6.61k-.flow 34M IJM7N3301.long-6.57k-.flow 4.0K IJM7N3301.flow.files
>
> # OUTPUT FROM COMMAND: mothur \> shhh.flows(file=IJM7N3301.flow.files, processors=8, lookup=…/…/mothur/LookUp\_Titanium.pat)
>
> # 8.5M IJM7N3301.long-6.82k-.shhh.qual 2.7M IJM7N3301.long-6.82k-.shhh.fasta 172K IJM7N3301.long-6.82k-.shhh.names 6.7M IJM7N3301.long-6.82k-.shhh.counts 184K IJM7N3301.long-6.82k-.shhh.groups 2.0M IJM7N3301.long-13.8k-.shhh.qual 616K IJM7N3301.long-13.8k-.shhh.fasta 224K IJM7N3301.long-13.8k-.shhh.names 8.7M IJM7N3301.long-13.8k-.shhh.counts 376K IJM7N3301.long-13.8k-.shhh.groups 13M IJM7N3301.shhh.fasta du: cannot access ‘IJM7N3301.shhh.namesi’: No such file or directory
>
> # OUTPUT FROM COMMAND …/…/mothur/mothur “#trim.seqs(fasta=IJM7N3301.shhh.fasta, name=IJM7N3301.shhh.names, oligos=IJM7N3301.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T, processors=8)”
> 0 IJM7N3301.shhh.trim.names
> 1.8M IJM7N3301.shhh.scrap.names
> 0 IJM7N3301.shhh.groups
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [May 21, 2014, 3:10am UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/5 "2014-05-21T03:10:58Z")
Can you run this…
cut -f 1 -d " " IJM7N3301.scrap.flow | cut -f 2 -d “|” | sort | uniq -c
And post the results?
---
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 21, 2014, 4:13pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/6 "2014-05-21T16:13:39Z")
cut -f 1 -d " " IJM7N3301.scrap.flow | cut -f 2 -d “|” | sort | uniq -c
> 1 1670
> 1 b
> 530 bf
> 174 f
> 25 h
> 80 l
> 11 lbf
> 2 lf
---
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 21, 2014, 7:04pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/7 "2014-05-21T19:04:02Z")
I recently got feedback on the correct primer sets (that I previously reverse engineered) so I reprocessed and got this:
cut -f 1 -d " " IJM7N3301.scrap.flow | cut -f 2 -d “|” | sort | uniq -c
> 1 1670
> 1 b
> 467 bf
> 2606 f
> 25 h
> 82 l
> 7 lbf
> 4 lf
everything still going to scrap:
> # ===================================================================== OUTPUT FROM COMMAND trim.seqs(fasta=IJM7N3301.shhh.fasta, name=IJM7N3301.shhh.names, oligos=schloss.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T, processors=8)
>
> 0 IJM7N3301.shhh.trim.fasta
> 13M IJM7N3301.shhh.scrap.fasta
> 0 IJM7N3301.shhh.trim.names
> 1.8M IJM7N3301.shhh.scrap.names
> 0 IJM7N3301.shhh.groups
New primer/mid file:
> forward CCGTCAATTCMTTTRAGT
> barcode TTCTCAAC mock1
> barcode TCACAC mock2
> barcode AACCTGGC mock3
> barcode AGACAC soil1
> barcode ACGCGC soil2
> barcode ACTCAC soil3
> barcode TCTCCGTC human1
> barcode TGAAGC human2
> barcode TGGTGAAC human3
> barcode AGACCTC mouse1
> barcode ATACCAC mouse2
> barcode TCGCGGC mouse3
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [May 21, 2014, 7:42pm UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/8 "2014-05-21T19:42:21Z")
ah, sorry - when you run shhh.flows you also need order=B
---
### Author:  [@JJ17](https://forum.mothur.org/u/JJ17)
#### Post date: [May 22, 2014, 12:55am UTC](https://forum.mothur.org/t/trim-flows-on-flx-or-junior-3-0-data/1853/9 "2014-05-22T00:55:16Z")
I think I’m good. Thanks! Btw the doc page on shhh.flows doesn’t list order= as an option…at least not in the example commands. I think there is a typo on that page as it’s trim.flows and not shhh.flows:
```plaintext
trim.flows(fasta=GQY1XT001.flow, order=A)
```
> **[shhh.flows](http://mothur.org/wiki/Shhh.flows)**
>
> The website that supports the mothur software program - one of the most widely used tools for analyzing 16S rRNA gene sequence data. Step inside to learn how to use the software, get help, and join our community!
My results below:
> # ===================================================================== OUTPUT FROM COMMAND trim.seqs(fasta=IJM7N3301.shhh.fasta, name=IJM7N3301.shhh.names, oligos=schloss.oligos, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, flip=T, processors=8)
>
> 12M IJM7N3301.shhh.trim.fasta
> 8.0K IJM7N3301.shhh.scrap.fasta
> 1.8M IJM7N3301.shhh.trim.names
> 4.0K IJM7N3301.shhh.scrap.names
> 2.1M IJM7N3301.shhh.groups
cut -f 1 -d " " IJM7N3301.scrap.flow | cut -f 2 -d “|” | sort | uniq -c
> 1 1670
> 1 b
> 467 bf
> 2606 f
> 25 h
> 82 l
> 7 lbf
> 4 lf
mothur \> summary.seqs(fasta=IJM7N3301.shhh.trim.fasta, name=IJM7N3301.shhh.trim.names)
> Using 1 processors.
> Start End NBases Ambigs Polymer NumSeqs
> Minimum: 1 450 450 0 4 1
> 2.5%-tile: 1 525 525 0 4 2532
> 25%-tile: 1 558 558 0 5 25319
> Median: 1 567 567 0 5 50638
> 75%-tile: 1 579 579 0 5 75957
> 97.5%-tile: 1 597 597 0 6 98744
> Maximum: 1 631 631 0 8 101275
> Mean: 1 566.1 566.1 0 5.00191
>
> # of unique seqs: 21131
>
> total # of seqs: 101275
> Output File Names:
> IJM7N3301.shhh.trim.summary