STAR Script Add a `//` Error

Question

[Y]

My script has a couple of variables pre-defined:

# Path to the genome FASTA file from Illumina iGenomes For Danio rerio (in this case):
Genome_FASTA="path/to/genome.fa"

# Path to the annotation GTF file:
Annotation_GTF="path/to/Danio_rerio.GRCz11.110.chr.gtf"

# Path to the input directory containing FASTQ files:
Input_Directory="/path/to/FQ_Folder"

# Output directory for alignment results:
Output_Directory="Path/to/Realigned_To_Ensembl_Folder_With_BAMs"

# Created the reference genome directory:
Reference="/path/to/STAR_Reference_Created_Previously"

The main script:

# Loop through each pair of paired FASTQ files in the input directory and subdirectories
for forward_file in "${Input_Directory}"/*_R1.fq; do
    # Extract the file name without extension
    file_name=$(basename "${forward_file}" _R1.fq)

    # Path to the corresponding reverse FASTQ file
    reverse_file="${forward_file/_R1/_R2}"

    echo "Forward File: ${forward_file}"
    echo "Reverse File: ${reverse_file}"
    echo "Output Directory: ${Output_Directory}"

    # Create a unique temporary directory for this sample
    TMPDIR="${Output_Directory}/${file_name}___STARtmp"

    echo "The temporary directory is: ${TMPDIR}"

    # Change working directory to the output directory
    cd "${Output_Directory}"

    echo "Made temporary directory: ${TMPDIR}"

    echo "Output Directory + Prefix: ${Output_Directory}/${file_name}___"

    # Run STAR alignment
    STAR \
        --genomeDir "${Reference}" \
        --readFilesIn "${forward_file}" "${reverse_file}" \
        --outFileNamePrefix "${Output_Directory}/${file_name}___" \
        --runThreadN "${Number_Of_Threads}" \
        --genomeLoad NoSharedMemory \
        --outSAMtype BAM SortedByCoordinate \
        --outTmpDir "${TMPDIR}" \
        --outStd Log \
        --outSAMunmapped Within \
        --outSAMattributes Standard

    # Index the generated BAM file
    samtools index "${Output_Directory}/${file_name}___Aligned.sortedByCoord.out.bam"

    rm -rf "$TMPDIR"/*

done

My error is:

Forward File: path/to/Sample-1_R1.fq
Reverse File: path/to/Sample-1_R2.fq
Output Directory: Path/to/Realigned_To_Ensembl_Folder_With_BAMs
Made temporary directory: /Path/to/Realigned_To_Ensembl_Folder_With_BAMs/Sample-1___STARtmp
Output Directory + Prefix: /Path/to/Realigned_To_Ensembl_Folder_With_BAMs/Sample-1___
Aug 13 16:50:22 ..... started STAR run
Aug 13 16:50:22 ..... loading genome
Aug 13 16:50:28 ..... started mapping
Aug 13 17:00:20 ..... finished mapping
Aug 13 17:00:21 ..... started sorting BAM

EXITING because of FATAL ERROR: failed reading from temporary file: /Path/to/Realigned_To_Ensembl_Folder_With_BAMs/Sample-1___STARtmp//BAMsort/7/48

It keeps adding a // after the the temporary directory.

I first posted it on Github here. I am not sure it was allowed on the STAR GitHub I added in some brackets {} and continued to try and modify it but I still cannot seem to get it to work. If the post is not allowed here I can take it down. I just want to resolve the issue with this script.

Answer 1

[petebio]

I don't think that the // is the problem here.

Rather, it appears that your script is trying to write to a root directory called /path which may not exists or that you may not have permission to write to.

Could you check if each of the directories + files specified by your pre-defined variables actually exist and are writable? Particularly the input directory and reference variables which both contain /path

[Y]

I did and it seems to be working currently (though the script is still running). Thank you.

[Ram]

Don't delete your post. I've moved petebio's comment to an answer. Please accept it to provide closure to the post.

[Y]

Ok okay it seemed that it was an issue with an older module of STAR not an issue with the script.