hisat2 error

Question

[loicborcard01]

Hi,

I am running my alignment on our cluster and I am getting this error on 5 fastq files

Warning: Same mate file "trimmed/SRR9695027_1.trimmed.fastq" appears as argument to both -1 and -2

Error while flushing and closing output

I checked my script and it doesn't look like I am inputting twice the same fastq file

this is my input parallel -j 20 'hisat2 -x ~/Projects/sheep/ref/Ovis_aries_rambouillet.Oar_rambouillet_v1.0.dna.toplevel.fa -1 trimmed/{}_1.trimmed.fastq -2 trimmed/{}_2.trimmed.fastq -S bam/{}.sam' :::: raw_data/sra_lung.txt

where sra_lung.txt contains sra ids (e.g SRR9695027)

thanks a lot

[Ram]

Use parallel's --dry-run option to see how your commands are being translated. That'll help you fix them.

[loicborcard01]

I did and it looks correct. hisat2 -x ~/Projects/sheep/ref/Ovis_aries_rambouillet.Oar_rambouillet_v1.0.dna.toplevel.fa -1 trimmed/SRR9695029_1.trimmed.fastq -2 trimmed/SRR9695029_2.trimmed.fastq -S bam/SRR9695029.sam

[Ram]

Your command looks fine to me too. Try wrapping the parallel command in a shell script and use set +x as the first line in that shell script - this will have the shell print every command it executes. IMO what you're seeing is very odd and should not happen.

cat my_shell_script.bash # the three lines below are the contents of your shell script
#!/bin/bash
set +x
parallel -j 20 'hisat2 -x ~/Projects/sheep/ref/Ovis_aries_rambouillet.Oar_rambouillet_v1.0.dna.toplevel.fa -1 trimmed/{}_1.trimmed.fastq -2 trimmed/{}_2.trimmed.fastq -S bam/{}.sam' :::: raw_data/sra_lung.txt

Now, run the shell script like so:

bash my_shell_script.bash 2>log.err | tee log.out #this should print every command being executed and save both STDOUT and STDERR to log files

[loicborcard01]

I have the output of my command from Slurm (HPC management software) if that helps and it seems that only the last fastq file caused this problem. Could this be due to a lack of memory or bad parallelization settings?

thanks for the help

> Warning: Same mate file "trimmed/SRR9695027_1.trimmed.fastq" appears as argument to both -1 and -2
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): hisat2-align died with signal 6 (ABRT) 
Warning: Same mate file "trimmed/SRR9695026_1.trimmed.fastq" appears as argument to both -1 and -2
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): hisat2-align died with signal 6 (ABRT) 
Warning: Same mate file "trimmed/SRR9695029_1.trimmed.fastq" appears as argument to both -1 and -2
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): hisat2-align died with signal 6 (ABRT) 
Warning: Same mate file "trimmed/SRR9695028_1.trimmed.fastq" appears as argument to both -1 and -2
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): hisat2-align died with signal 6 (ABRT) 
Warning: Same mate file "trimmed/SRR9695025_1.trimmed.fastq" appears as argument to both -1 and -2
Warning: skipping mate #1 of read 'SRR9695025.11135 11135 length=151' because length (0) <= # seed mismatches (0)
Warning: skipping mate #2 of read 'SRR9695025.11135 11135 length=151' because length (0) <= # seed mismatches (0)
Warning: skipping mate #1 of read 'SRR9695025.11135 11135 length=151' because it was < 2 characters long
Warning: skipping mate #2 of read 'SRR9695025.11135 11135 length=151' because it was < 2 characters long

[Ram]

There is a mismatch between what you're saying and what the content you've pasted above is saying. The content indicates that there's a problem with every FASTQ pair.