# Make.contigs returning empty sability.trim.contigs.fasta

**URL:** <https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064>\
**Category:** Commands in mothur\
**Created:** [August 14, 2019, 1:37am UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064 "2019-08-14T01:37:58Z")\
**Posts on this page:** 5\
**Page:** 1

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**Author:** ![napaqtuk](https://avatars.discourse-cdn.com/v4/letter/n/ac91a4/32.png) [@napaqtuk](https://forum.mothur.org/u/napaqtuk)\
**Post date:** [August 14, 2019, 1:37am UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064/1 "2019-08-14T01:37:58Z")

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Using mothur v.1.42.1

I am using the Miseq SOP. I am able to make stability files using make.file. However, when I run make.contigs and then check the files with summary.seqs, I get the same error:

#[ERROR]: stability.trim.contigs.fasta is blank. Please correct. Error in reading your fastafile, at position -1. Blank name.

Here is the log from my last run:

```auto
mothur > make.contigs(file=stability.files, processors=20, oligos=OligoFluridoneRev3.txt)

Using 20 processors.

>>>>>	Processing file pair Undetermined_S0_L001_I1_001.fastq - Undetermined_S0_L001_I2_001.fastq (files 1 of 2)	<<<<<
Making contigs...
Done.

It took 775 secs to assemble 10351587 reads.

>>>>>	Processing file pair Undetermined_S0_L001_R1_001.fastq - Undetermined_S0_L001_R2_001.fastq (files 2 of 2)	<<<<<
Making contigs...
Done.

It took 6324 secs to assemble 10351587 reads.

It took 7104 secs to process 20703174 sequences.

Output File Names: 
stability.trim.contigs.fasta
stability.trim.contigs.qual
stability.scrap.contigs.fasta
stability.scrap.contigs.qual
stability.contigs.report

mothur > summary.seqs(fasta=stability.trim.contigs.fasta)

Using 20 processors.
[ERROR]: stability.trim.contigs.fasta is blank. Please correct.
Error in reading your fastafile, at position -1. Blank name.

```

The first three lines of my oligo file looks like this:  
BARCODE|GACACAGT|CTAGGTGA|C5.1|  
|BARCODE|TTGCTTGG|ACACGGTT|C5.2|  
|BARCODE|TATGGCAC|GGACCTAT|C5.3|

Where each | is a tab. I have tried several iterations of the oligo file, using full oligos, or not using the reverse complement of the reverse barcode, or not including the oligo file at all. I get the same error for most runs.

My input files at the start are:  
Undetermined\_S0\_L001\_I1\_001.fastq  
Undetermined\_S0\_L001\_I2\_001.fastq  
Undetermined\_S0\_L001\_R1\_001.fastq  
Undetermined\_S0\_L001\_R2\_001.fastq

I appreciate your help.

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [August 15, 2019, 2:30pm UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064/2 "2019-08-15T14:30:38Z")

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Hey Kat,

Hmmm. I suspect that your Undetermined\_S0\_L001\_I1\_001.fastq and Undetermined\_S0\_L001\_I2\_001.fastq files contain sequences that are only 8 nt long and your other files have sequences that are 251 nt long. I’m pretty sure the “I files” contain the index/barcode sequences. Those won’t show up in the “R files” and `make.contigs` won’t find them either putting everything in the scrap files.

Could you try something like this?

```auto
make.contigs(ffastq=Undetermined_S0_L001_R1_001.fastq, rfastq=Undetermined_S0_L001_R2_001.fastq, findex=Undetermined_S0_L001_I1_001.fastq, rindex=Undetermined_S0_L001_I2_001.fastq, oligos=OligoFluridoneRev3.txt)

```

Pat

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<div class="post-metadata">

**Author:** ![napaqtuk](https://avatars.discourse-cdn.com/v4/letter/n/ac91a4/32.png) [@napaqtuk](https://forum.mothur.org/u/napaqtuk)\
**Post date:** [August 15, 2019, 9:57pm UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064/3 "2019-08-15T21:57:51Z")

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Oh, I had such high hopes. I ran your suggested code, did the summary.seqs, and got this error:  
Stability.trim.contigs.fasta is blank. Please correct. Error in reading your fastafile, at position -1. Blank name.

You are correct though, the I1/I2 files contain sequences that are only 8 nt long and the R1/R2 files have long sequences. I checked my primer files and barcode files, and can’t find the sequences contained in the index files in either.

For kicks, I ran your code without the I1/I2 files. Sadly that didn’t work either.

Thanks, Kat

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [August 16, 2019, 3:29pm UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064/4 "2019-08-16T15:29:44Z")

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Hmmm. Could you email me the I1/I2 files along with your oligos file? It might be a problem with reverse complements or something like that.

Generally, the sequencer does the parsing for us for cases like this and would split the R1/R2 file according to the barcode.

Pat

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<div class="post-metadata">

**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [August 26, 2019, 3:29pm UTC](https://forum.mothur.org/t/make-contigs-returning-empty-sability-trim-contigs-fasta/20064/5 "2019-08-26T15:29:50Z")

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