STAR alignment EXITING because of FATAL ERROR: could not create output file

Question

[gnomee]

I am trying to align RNA-Seq data using STAR. However, STAR does not start the alignment, but exits with the following error:

   EXITING because of FATAL ERROR: could not create output file: /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE_L001_Log.out
Check if the path /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE_L001_ exists and you have permissions to write there

However, I do have permissions for writing in the directory.

My command was the following:

STAR --genomeDir hg_GRCh38/indexes/STAR/STAR_2.5.3a_Gencode32_100bp_completep \
    --sjdbGTFfile hg_GRCh38/gencode32/gencode.v32.annotation.gtf \
    --readFilesIn 65312-RNA-FFPE_S12_L001_R1_001_trimmed.fastq.gz 65312-RNA-FFPE_S12_L001_R1_001_trimmed.fastq.gz\
    --outFileNamePrefix /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE_L001_ \
    --runThreadN 12 --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif \
    --readFilesCommand zcat --outTmpDir /home/c479e/ANTCoN_analysis

I already tried lowering --runThreadN to 6, and setting ulimit -n to even 100k, but I still get the same error. Does anybody know what could be going wrong here?

Answer 1

[caggtaagtat]

Hi, I think the argument --outFileNamePrefix wants to have a directory as an input, where the results of STAR will be saved.

If you just do this STAR run, I would first create the output directory

mkdir 65312-RNA-FFPE

and then execute STAR with this parameter:

--outFileNamePrefix /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE/

I guess you have paired end reads and the second file should have an R2 in the name? Is this just a spelling error here or also in the script?

--readFilesIn 65312-RNA-FFPE_S12_L001_R1_001_trimmed.fastq.gz 65312-RNA-FFPE_S12_L001_R1_001_trimmed.fastq.gz

should be?

--readFilesIn 65312-RNA-FFPE_S12_L001_R1_001_trimmed.fastq.gz 65312-RNA-FFPE_S12_L001_R2_001_trimmed.fastq.gz

[gnomee]

This solved it! I changed the --outFileNamePrefix to /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE/ as you suggested, and now it runs fine. I thought it was also possible to specify the first bit of the file name with --outFileNamePrefix. The R2 was a spelling error in the post, it is correct in my code. Thank you!

Answer 2

[Thibault D.]

Hi,

STAR error is pretty clear to me:

Check if the path /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE_L001_ exists and you have permissions to write there

Just run: touch /home/c479e/ANTCoN_analysis/STAR/65312-RNA-FFPE_L001_

And if you get errors while touch-ing, try: mkdir --parents /home/c479e/ANTCoN_analysis/STAR/

I often forget to create output directories too, these commands always fix the error or help me to understand real error beneath.

[gnomee]

The touch command worked, which was what puzzled me in he first place.