I have been using bowtie2 to align 150bp illumina miseq reads to a reference genome and would like to output unmapped reads to a separate file for further investigation. I have tried using the command below (all and local are selected for my application)
bowtie2 -a --local -x index -1 R1.fastq.gz -2 R2.fastq.gz -S out.sam --un-conc unmapped.fq
I get a sam file and 2 unmapped reads fastq files which contain the exact number of unmapped reads I expect to from the Bowtie2 output. My concern is when I run the above command I get multiple warnings as below
Use of uninitialized value $bt2_args[4] in join or string at /Users/dir/Documents/anaconda3/bin/bowtie2 line 423.
Use of uninitialized value $bt2_args[5] in join or string at /Users/dir/Documents/anaconda3/bin/bowtie2 line 423.
Use of uninitialized value in exists at /Users/dir/Documents/anaconda3/bin/bowtie2 line 81.
Use of uninitialized value in exists at /Users/dir/Documents/anaconda3/bin/bowtie2 line 81.
Use of uninitialized value $bt2_args[4] in join or string at /Users/dir/Documents/anaconda3/bin/bowtie2 line 459.
Use of uninitialized value $bt2_args[5] in join or string at /Users/dir/Documents/anaconda3/bin/bowtie2 line 459.
The sam file I get is the same as the one I get when I run the command without --un-conc. I have checked the lines in the conda script but they don't mean enough to me to be able to work out what parameters I am missing. I have tried moving the --un-conc command to before the -x and I get the exact same warnings and output. I get no errors if I remove the -un-conc command so it is this which is causing the warnings to occur.
Does anyone know why this is happening especially as it doesn't appear that the warnings are causing any issues with the output files that I can see? Is there anything I should be checking to assure myself that the files are as they should be?
Should I be worried or can I ignore them (although I would like to figure out what is causing this!)?
Thanks in advance for your help
Perfect - thanks very much. In hindsight I should have checked some links further down on google as I clearly see this now. Could have saved myself some time!