# empty fasta file
**URL:** https://forum.mothur.org/t/empty-fasta-file/2662
**Category:** Commands in mothur
**Created:** [January 19, 2016, 1:07am UTC](https://forum.mothur.org/t/empty-fasta-file/2662 "2016-01-19T01:07:53Z")
**Posts on this page:** 5
**Page:** 1
### Author:  [@H\_Sapers](https://forum.mothur.org/u/H_Sapers)
#### Post date: [January 19, 2016, 1:07am UTC](https://forum.mothur.org/t/empty-fasta-file/2662/1 "2016-01-19T01:07:53Z")
I’ve been having trouble processing one set of sequence data (Titanium GS flex +). After extracting using sffinfo, I’m running trim.flows(flow=iron.flow, oligos=iron.oligos, order=B, pdiffs=5, bdiffs=2, maxhomop=8, minflows=200, maxflows=1050, fasta=T). (I’ve tried various minflows and maxflows values). Everything seems to go into scrap.flows. I’ve opened scrap.flows and sequences are thrown out at the barcode and primer level. I’ve opened up the fasta file and checked the barcode and primer in the oligos file, but can’t seem to figure out why they’re being thrown out.
Thanks!
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### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [January 19, 2016, 2:19pm UTC](https://forum.mothur.org/t/empty-fasta-file/2662/2 "2016-01-19T14:19:47Z")
Can you post the primers/barcdoes you are using along with ~10 fasta sequences?
Once we get this figured out, you’re going to need to use minflows and maxflows values that are the same.
Pat
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### Author:  [@H\_Sapers](https://forum.mothur.org/u/H_Sapers)
#### Post date: [January 23, 2016, 4:06pm UTC](https://forum.mothur.org/t/empty-fasta-file/2662/3 "2016-01-23T16:06:48Z")
Thanks Pat!
I’m using 27Fmod (5’-AGRGTTTGATCMTGGCTCAG-3’) and reverse 519 (5’-TTACCGCGGCTGCTGGCAC-3’)
Here is the oligos file
#forward AGRGTTTGATCMTGGCTCAG
forward AGGGTTTGATCATGGCTCAG
barcode AATAGCGG Green
barcode AATACGCC Mixed
and a few fasta sequences
> IH7IBDS01C3JPF xy=1153\_1777
> AATAGCGGAGTAGTTTGATCATGGCTCAGATTGACGCTGGCGCAGGCTTAACACATGCAAGTCGAGCGGAAACGNTGGTANCTTGCTACCAGGCGTCGAGCGGCGGACGGTNAGTATGCTTAGGATCTGCCTAGTAGTGGGGATACGGCCCGGGAACTCGGATTAANACCGCATACGCCGTACGGGGAAGCAGGGATCTTCGACCTGCGCTATTAGATGAGCCTAAGTCGGATAGCTTGTTGGTGGGTAATGCCTACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCA
> IH7IBDS01C4Q5G xy=1167\_738
> AATACGCCAGGGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAAACGATGGTAGCTTGCTACCAGGCGTCGAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCCAGTAGTGGGGAGTAGCCCGGGGAACGTACGGATTAATACCGCATACGCCCTTTTCGGGGTAAAGGACAGGGGAGTCTTCGGACCTTGCGCTATTGGATGAGCCTAAGTCGGATTAGCTTGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGGCAGCAGTGGGGAAGTCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTAGGGTCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
> IH7IBDS01C7XH6 xy=1203\_1484
> AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGAGATCTTCGGATCTAGCGGCGGACGGGTGAGTAACGCGTGGGAATATGCCCTTCTCTACGGAATAGCCTCGGGAAACTGGGAGTAATACCGTATACGCCTCGGGAAGGATTATCGAGAGATAGCCGCGTGATAGTAGTGTGGGTAGTGCTACCAGCGACGATCATAGCTGGTTTGTAGAGGATGATCAGCCACACTGGGACGTGAGACACGGCCCACGACTCCTACGGGAGGCGAGCAGTGGGGAAGTCTTAGACAATAGGGGGAAACCCGTAGATCTAGCCATGCCGCGTGAGCGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCAGCTGGGAAGATAATGACGGTACCAGCAGAAGAAGCCCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAAT
> IH7IBDS01D7NBE xy=1610\_824
> AATAGCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAACACATGCAAGTCGAGGGGTAACAGGAGAGCTTGCTCTTGCTGACGACCGCGCACGGGTGCGTAACGCGTATACAATCTACCTATTACTAAGAGATAGCCCACGAGAAATTTAGGTATTAATAATTTTATTGGTATTACAATTTGGTCATCAAGTTAGTAATTAAAGGTTACGGTAATAGATGAGTATGCGTCCTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCTACGATAGGTAGGGGTCCGTGAGAGGGAGATCCCCCACCACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGACAATGGGAGCAATCCTGATCCAGCCATGCCGCGTGCAGGAAGACTGCCCTATGGGTTGTAAACTGCTTTTATACAGGAAGAAAAAGGTGTACGTGTACACTGTTGACGGTACTGTAAGAATAAGGATCGGCTAACTCCGTGCCAG
> IH7IBDS01AN2V5 xy=157\_531
> AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGCGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGAAAGATTTATCGGTAAGGATTGGCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTTAGGGTCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACT
> IH7IBDS01CNS9B xy=974\_845
> AATACGCCAGAGTTTGATCATGGCTCAGAATGAACGCTGGCGGTAGGCTTAACACATGCAAGTCGGACGGAAACGATAGAGAAGCTTGCTTTTCTAGGCGTCGAGTGGCGGACGGGTGAGTAACGCGTGGGAATCTACCCTATAGTTGGGGACAACGTATGGAAACGTACGCTAATACCGAATATGCTCTACGGAGTAAAGGAGCCCTCTTCCTGAAAGGTTTCGCTATAGGATGAGCCCGCGTGAGATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATCTCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGACGAAAGTCTGATGCAGCCATACCGCGTGTGTGAAGAAGGCCCGAGGGTTGTAAAGCACTTTCAATTGTGAGGAAGGTAGTGTAGTTAATACCTGCATTGCTTGACGTTAACTTTAGAAGAAGCACCGGCTAACTCTGTGCC
> IH7IBDS01DS6QB xy=1445\_1985
> AATACGCCAGGGTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGGCGGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGGAAAGGATTTATCGGTAAGGATTGGCCCGCGTAAGATTAGATAATTGGTGGGGTAAGTGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGTAGGATGATCAGCAACACTGGGGACTGAGACACGGCCCAGACTCCTACGGGAGGGCAGCAGTGGGGAAGTCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTAGGGTCGTAAAGCACTTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
> IH7IBDS01CGMZ0 xy=892\_2014
> AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCTTCGGGTGAGCGGCGGACGGGTTAGTAACGCGTGGGAACGTACCCTTTTCTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATAAGCCCTTCGGGGGAAAGATTTATCGGGAAAGGATCGGCCCGCGTTAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTATAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGTCTTAGGATCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
> IH7IBDS01CNVY2 xy=975\_268
> AATAGCGGAGGGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAACACATGCAAGTCGAGGGGTAACAGGAGAGCTTGCTCTTGCTGACGACCGGCGCGACGGGTGCGTAACGCGTATACAATCTACCTATTACTAAGAGATACGCCCAGAGTAAATTTGGATTAATATTTTATGGTATTACAATTTGGCATCAAGTTGTAAATTAAAGGTTACGGTAATAGATGAGTATGCGTCCTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCTACGATAGGGTAGGGGTCCTGA
> IH7IBDS01DE09S xy=1284\_1182
> AATACGCCAGGGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGGCGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGTAAAGGATTGGCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGAATGAAGGGCCTTTAGGGTCGTAAAAGCACTTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCAGCGCGGTAATT
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### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [January 25, 2016, 1:09pm UTC](https://forum.mothur.org/t/empty-fasta-file/2662/4 "2016-01-25T13:09:07Z")
A couple things…
First, you want this…
```nohighlight
forward AGRGTTTGATCMTGGCTCAG
#forward AGGGTTTGATCATGGCTCAG
barcode AATAGCGG Green
barcode AATACGCC Mixed
```
Second, are you positive that you have flow order B? What happens if you try A with minflows/maxflows=450? The reads you posted aren’t super long (~550 nt long?), which makes me think you likely have A.
Third, if you are running mothur on mac/linux can you send the output of running this on your scrap.flow file?
```nohighlight
cut -f 1 -d " " *scrap.flow | cut -f 2 -d "|" | sort | uniq -c
```
Pat
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### Author:  [@H\_Sapers](https://forum.mothur.org/u/H_Sapers)
#### Post date: [February 3, 2016, 4:38pm UTC](https://forum.mothur.org/t/empty-fasta-file/2662/5 "2016-02-03T16:38:25Z")
Thanks Pat
Changing the order seemed to do the trick… I was told the sequencing was done with the new chemistry on a GS flx + platform, but this indeed may not be the case…