How to align zebrafish RNA-seq data against a reference genome with splice sites using hisat2?

Question

[tara]

Hey,

I want to align RNA-seq fastq files against a reference genome with splice sites using hisat2. First, I downloaded a reference genome and built an index using the hisat2-build function.

wget ftp://ftp.ensembl.org/pub/release-102/fasta/danio_rerio/dna/Danio_rerio.GRCz11.dna.primary_assembly.fa.gz
gzip -d Danio_rerio.GRCz11.dna.primary_assembly.fa.gz
mv Danio_rerio.GRCz11.dna.primary_assembly.fa genome.fa
hisat2-build -p 16 genome.fa genome

Then I downloaded the corresponding gtf file and created the hisat2 specific splice sites file:

wget ftp://ftp.ensembl.org/pub/release-102/gtf/danio_rerio/Danio_rerio.GRCz11.102.gtf.gz
gzip -d Danio_rerio.GRCz11.102.gtf.gz
mv Danio_rerio.GRCz11.102.gtf genome.gtf 
hisat2_extract_splice_sites.py genome.gtf > genome.txt

Finally, I tried to align the paired end fastq files against the index.

hisat2 --dta --known-splicesite-infile genome.txt -x genome -1 fastq/mut_1_1.fastq.gz -2 fastq/mut_1_2.fastq.gz > hisat2/mut_1.sam

The output file is generated. But when it reaches a size of 4.0 GB, there is always this error message:

Error while flushing and closing output  
terminate called after throwing an instance of 'int' 
Aborted (core dumped)
(ERR): hisat2-align exited with value 134

I searched what this error could mean, and it might be due to too less memory space. I checked the storage, and the Hard Drive has 75 GB of free space. I have all my files on an external media and there are nearly 400 GB space left. This should be enough.

Can you help me in understanding and solving the error?

[ATpoint]

Is it intentional that the splice file ends with .ss but you feed a .txt to hisat?

hisat2_extract_splice_sites.py genome.gtf > genome.ss
hisat2 --dta --known-splicesite-infile => genome.txt <= right there

[tara]

Thanks for this hint. I corrected the code above. I was wondering if the splice site file is occuring the error and tried to align the fastq files against the reference genome without the file:

hisat2 --dta -x genome -1 fastq/mut_1_1.fastq.gz -2 fastq/mut_1_2.fastq.gz > hisat2/mut_1.sam

But this leads to the same error like above.