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

Commands in mothur
1

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:

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.

2

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?

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

3

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

4

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

5

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