I am trying to run Bowtie2 alignment in a Nextflow pipeline, but I’m encountering an error where Bowtie2 can’t find the index files. The error message indicates that "reference_index.1.bt2" does not exist or is not a Bowtie 2 index.
Here is my code.
// Define the process for Bowtie2 alignment
process bowtie2 {
input:
path bowtie2_out
path trimmomatic_out
output:
path "${params.outputDir8}/out.sam"
script:
"""
baseName=$(basename $(find ${bowtie2_out} -name '*.bt2' | head -n 1) | sed 's/\.[0-9]*\.bt2$//')
indexPath="${bowtie2_out}/${baseName}"
bowtie2 -x ${indexPath}
-1 ${trimmomatic_out}/output_1P.fq
-2 ${trimmomatic_out}/output_2P.fq
-S ${params.outputDir8}/out.sam
"""
}
I am trying to dynamically extract the base name of the Bowtie2 reference index and use it for the -x option in the bowtie2 command. However, the command error i get is:
Command error:
Index path is: reference_index.1.bt2 reference_index.2.bt2 reference_index.3.bt2 reference_index.4.bt2 reference_index.rev.1.bt2 reference_index.rev.2.bt2/reference_index
(ERR): "reference_index.1.bt2" does not exist or is not a Bowtie 2 index
Exiting now ...
The complete error message that I got is:
ERROR ~ Error executing process > 'bowtie2'
Caused by:
Process `bowtie2` terminated with an error exit status (255)
Command executed:
baseName=$(basename $(find reference_index.1.bt2 reference_index.2.bt2 reference_index.3.bt2 reference_index.4.bt2 reference_index.rev.1.bt2 reference_index.rev.2.bt2 -name '*.bt2' | head -n 1) | sed 's/.[0-9]*.bt2$//')
indexPath="reference_index.1.bt2 reference_index.2.bt2 reference_index.3.bt2 reference_index.4.bt2 reference_index.rev.1.bt2 reference_index.rev.2.bt2/${baseName}"
echo "Index path is: ${indexPath}"
bowtie2 -x ${indexPath} -1 trimmomatic_out/output_1P.fq -2 trimmomatic_out/output_2P.fq -S bowtie2_out/out.sam
Command exit status:
255
Command output:
Index path is: reference_index.1.bt2 reference_index.2.bt2 reference_index.3.bt2 reference_index.4.bt2 reference_index.rev.1.bt2 reference_index.rev.2.bt2/reference_index
Command error:
Index path is: reference_index.1.bt2 reference_index.2.bt2 reference_index.3.bt2 reference_index.4.bt2 reference_index.rev.1.bt2 reference_index.rev.2.bt2/reference_index
(ERR): "reference_index.1.bt2" does not exist or is not a Bowtie 2 index
Exiting now ...
Work dir:
/mnt/d/somil_ILBS/work/work/b3/358c00e746858cc5c99a755b28eba2
Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`
-- Check '.nextflow.log' file for details
I have tried various methods to extract the base name from the .bt2 files, but it seems the problem lies in how the path is being constructed.How can I correctly construct the path to the Bowtie2 index file so that Bowtie2 recognizes it and proceeds with the alignment?
From the command executed, it looks like bowtie2_out is expecting a directory, but it is in fact just a list of files. My suggestion would be to modify the upstream genome indexing process to output these into a directory so that they can be readily used downstream, for example:
process bowtie2_build {
input:
path fasta
output:
path "bowtie2"
script:
"""
mkdir bowtie2
bowtie2-build \
--threads ${task.cpus} \
"${fasta}" \
"bowtie2/${fasta.baseName}"
"""
}
Then feed bowtie2_build.out into your bowtie2 process. Your refactored bowtie2 process should look something like:
process bowtie2 {
publishDir "${params.outputDir}/bowtie2", mode: 'copy'
input:
tuple val(sample), path(fastq1), path(fastq2)
path bowtie_index
output:
tuple val(sample), path("${sample}.sam")
script:
"""
indexPath=$(find -L . -name '*.rev.1.bt2' | sed 's/\.rev.1.bt2$//')
bowtie2 \
-x "${indexPath}" \
-1 "${fastq1}" \
-2 "${fastq2}" \
-S "${sample}.sam"
"""
}
Note that you can use the publishDir directive to direct your process outputs to your designated output directory. This ensures files are written to the process working directory (i.e. ./work). The Trimmomatic process should also output a tuple containing the FASTQ files.