Puzzling Bowtie2 Input Error -S flag
Entering edit mode
6 months ago
geneticatt ▴ 130

Hi all,

I've come across some puzzling Bowtie2 behavior. I was attempting to align paired-end reads to the mouse genome using the bowtie2-align command by calling the bowtie2 wrapper, as recommended. As you can see below, I pass my fastq files to the -1 and -2 flags by looping through a range of numbers and using those to access the indices of three arrays, one with the R1 fastq filenames, another with the R2 and a third with the output filename for expected bam output.

My mistake was to call the -S flag without following up with a filename. The error that Bowtie gave was the result of some very weird argument processing. It looks like when the -S flag is called incorrectly as I called it, the filename given to the -1 flag is appended to the filename given to -2 with a space between them. Needless to say, this was a confusing error to run into, given the discrepancy between my script and the error.

My script

module load bowtie2
module load samtools

# Get Project Directory
BASE_DIR=$( pwd | rev | cut -d'/' -f2- | rev )

# get trimmed reads
READS1=( ${BASE_DIR}/trimmed/*_1.fastq )
READS2=( ${BASE_DIR}/trimmed/*_2.fastq )
OUT=( $( echo "${READS1[@]}" | sed 's/_1.fastq/.bam/g' | xargs -n 1 basename ) )

# align reads to genome

for ((i=0;i<=END;i++))
    bowtie2 --phred33 -p 16 -k 10 -x ${BASE_DIR}/genome/mus_musculus -1 "${READS1[i]}" -2 "${READS2[i]}" -S | samtools view -@ 12 -u - | samtools sort -@ 12 -n -o ${BASE_DIR}/bowtie/${OUT[i]} -

You can see the improperly called -S right before the pipe to samtools.

The output

[user@server]$ sh bowtie.slurm
Error: 0 mate files/sequences were specified with -1, but 1
mate files/sequences were specified with -2.  The same number of mate files/
sequences must be specified with -1 and -2.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /opt/applications/bowtie2/2.4.2/bowtie2-align-s --wrapper basic-0 -p 16 -k 10 -x /path/to/genome/mus_musculus -S -1 -2 /path/to/trimmed/trimmed_SAMN08610886_2.fastq path/to/trimmed/trimmed_SAMN08610886_1.fastq
(ERR): bowtie2-align exited with value 1

I wanted to leave this here in case somebody else came across the same issue. I also am wondering if it's proper to report unhelpful error messages and weird output behavior to the tool maintainers.

Thanks for any input on this.

bash bowtie2 alignment shell • 549 views
Entering edit mode

Is there an open question here or you are just reporting your mistake?

Entering edit mode

I have resolved the issue, but I am also wondering if this is considered a bug that needs reporting.

Entering edit mode

I don't know if this would be considered a bug. If you used the -S flag then you should have provided a file name. bowtie is not aware of what you are doing outside its command line. Since you are piping the output directly into samtools you don't need that option.

Entering edit mode

Thanks for the reply. I'm aware that the option isn't needed, but the error is seemingly irrelevant and misleading. So my question is: Are misleading error messages considered bugs?


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