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STAR Genome index Error

I tried to run STAR command for RNAseq but I got the following error.

/home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate \
> > --genomeDir GRCh38.79.chrom1 \
> > --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
> > --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
        /home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate --genomeDir GRCh38.79.chrom1 --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf --sjdbOverhang 62
> > --sjdbOverhang 62
*****!!!!! WARNING: --genomeSAindexNbases 14 is too large for the genome size=248956422, which may cause seg-fault at the mapping step. Re-run genome generation with recommended --genomeSAindexNbases 12*****
Oct 03 18:27:25 ... starting to sort Suffix Array. This may take a long time...
Oct 03 18:27:26 ... sorting Suffix Array chunks and saving them to disk...
Killed
star
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updated by Ram Ram • written by Prasanna • Prasanna

How much memory does you machine have?

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written by ATpoint ATpoint

Agreed it's a memory problem, I'm wondering if using the appropriate genomeSAindexNbases value helps in this case.

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written by Trivas • Trivas

From the documentation

For small genomes, the parameter --genomeSAindexNbases must to be scaled down, with a typical value of min(14, log2(GenomeLength)/2 - 1). For example, for 1 megaBase genome, this is equal to 9, for 100 kiloBase genome, this is equal to 7.

Using the size provided in the error, the value this person should be using is 13.

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written by biofalconch biofalconch 
STAR --runMode genomeGenerate \
--genomeDir GRCh38.79.chrom1 \
--genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
--sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
--sjdbOverhang 62

This is the code i am using... What should i modify to prevent the error

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updated by Ram Ram • written by Prasanna • Prasanna

add the flag --genomeSAindexNbases 12

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written by biofalconch biofalconch 
[pshekar@login002 RNAseq]$ /home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate \
> --genomeDir GRCh38.79.chrom1 \
> --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
> --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
> --genomeSAindexNbases 12
        /home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate --genomeDir GRCh38.79.chrom1 --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf --genomeSAindexNbases 12
        STAR version: 2.7.11a   compiled: 2023-10-03T18:15:28-04:00 login002.palmetto.clemson.edu:/home/pshekar/RNAseq/STAR-2.7.11a/source
Oct 04 10:56:22 ..... started STAR run
Oct 04 10:56:22 ... starting to generate Genome files
Oct 04 10:56:25 ..... processing annotations GTF
Oct 04 10:56:26 ... starting to sort Suffix Array. This may take a long time...
Oct 04 10:56:27 ... sorting Suffix Array chunks and saving them to disk...
Killed
[pshekar@login002 RNAseq]$ --sjdbOverhang 62
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updated by GenoMax GenoMax • written by Prasanna • Prasanna

Why did you add it in the middle instead of at the end? Run this:

STAR --runMode genomeGenerate \
--genomeDir GRCh38.79.chrom1 \
--genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
--sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
--sjdbOverhang 62 \
--genomeSAindexNbases 12
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written by Ram Ram

Could you answer my question?

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written by ATpoint ATpoint

I have no idea... I am new to bio informatics.. I am sorry

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written by Prasanna • Prasanna

Ask the system administrator. From your prompt, it looks like you're on a login node. DO NOT RUN high memory/long-running jobs on a login node. Contact your sysadmin about requesting a long running high memory node to run your job.

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written by Ram Ram

Can you tell me how to find it

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written by Prasanna • Prasanna

I am using my college's cluster computer

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written by Prasanna • Prasanna

Are you using a compute node or just the login node? If you're using a compute node, how much RAM did you request for allocation?

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written by Ram Ram

I am using through online cluster

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written by Prasanna • Prasanna

What does that even mean? What is an "online cluster"?

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written by Ram Ram 
[pshekar@login002 RNAseq]$ /home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate \
> --genomeDir GRCh38.79.chrom1 \
> --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
> --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
> --genomeSAindexNbases 12
        /home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate --genomeDir GRCh38.79.chrom1 --genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa --sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf --genomeSAindexNbases 12
        STAR version: 2.7.11a   compiled: 2023-10-03T18:15:28-04:00 login002.palmetto.clemson.edu:/home/pshekar/RNAseq/STAR-2.7.11a/source
Oct 04 10:56:22 ..... started STAR run
Oct 04 10:56:22 ... starting to generate Genome files
Oct 04 10:56:25 ..... processing annotations GTF
Oct 04 10:56:26 ... starting to sort Suffix Array. This may take a long time...
Oct 04 10:56:27 ... sorting Suffix Array chunks and saving them to disk...
Killed
[pshekar@login002 RNAseq]$ --sjdbOverhang 62
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updated by GenoMax GenoMax • written by Prasanna • Prasanna

It still shows error

/home/pshekar/RNAseq/STAR-2.7.11a/source/STAR --runMode genomeGenerate \
--genomeDir GRCh38.79.chrom1 \
--genomeFastaFiles genome/Homo_sapiens.GRCh38.dna.chromosome.1.fa \
--sjdbGTFfile gtf/Homo_sapiens.GRCh38.79.chrom1.gtf \
--genomeSAindexNbases 12
--sjdbOverhang 62
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updated by GenoMax GenoMax • written by Prasanna • Prasanna

Do you have write permissions to genomeDir specified above. It appears that your process is killed right after it tries to write to the disk location.

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written by GenoMax GenoMax

I didn't write permissions for those... How do I do that

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written by Prasanna • Prasanna

If you execute touch GRCh38.79.chrom1/testfile do you get an error? If you do then you don't have write permissions to the directory.

This may be a good point to stop and familiarize yourself with unix command line, if you are not aware of unix file permissions. Simply issuing commands (without understanding the logic) is not a good way of doing analysis.

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updated by Ram Ram • written by GenoMax GenoMax

Can you please suggest how can I modify the code ... Thanks!

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written by Prasanna • Prasanna

Code itself looks fine. If you are trying to run this on a cluster without going through the job scheduler then it is possible that admins may have set something up to kill jobs that are run outside of the job scheduler. You will need to ask the local support if you are not familiar with how the cluster is setup.

Generally admins frown upon people running analysis jobs on login nodes (which is where you seem to be running this from). Jobs are meant to be run via a job scheduler (SLURM/SGE etc).

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written by GenoMax GenoMax

Your code had a backslash instead of a forward slash (GRCh38.79.chrom1\testfile - I fixed it) - the touch test will not yield useful results with that.

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written by Ram Ram

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