# I am getting this error when running a test data set for Lyve-SET on RHEL 7. Is this an MPileup error or something else?

This is the error that I am getting:

launch_varscan.pl: Creating a pileup /home/user/lyve-SET-1.1.4f/lambda/tmp/NC001416.fasta.wgsim.fastq.gz-reference.sorted.bam.mpileup
removed ‘/home/user/lyve-SET-1.1.4f/lambda/tmp/NC001416.fasta.wgsim.fastq.gz-reference.sorted.bam.1239.mpileup’
launch_varscan.pl: Running mpileup:
echo "NC_001416.1
" | xargs -P 1 -n 1 -I {} sh -c 'echo "MPileup on {}" >&2; samtools mpileup -f lambda/reference/reference.fasta
-Q 0 -B  --region "{}" /home/user/lyve-SET-1.1.4f/lambda/bam/NC001416.fasta.wgsim.fastq.gz-reference.sorted.bam >
/home/user/lyve-SET-1.1.4f/lambda/tmp/NC001416.fasta.wgsim.fastq.gz-reference.sorted.bam..mpileup'
MPileup on NC_001416.1
mpileup: unrecognized option '--region'
open: No such file or directory
[mpileup] failed to open NC_001416.1: No such file or directory
launch_varscan.pl: main::mpileup: ERROR with xargs and samtools mpileup
Stopped at /home/arahat/lyve-SET-1.1.4f/scripts/launch_varscan.pl line 106.
QSUB ERROR
31488
launch_set.pl: Schedule::SGELK::command: ERROR with command: Inappropriate ioctl for device
/usr/bin/perl /home/user/lyve-SET-1.1.4f/lambda/log/SGELK/qsub.2705.pl
Stopped at /home/user/lyve-SET-1.1.4f/scripts/../lib/Schedule/SGELK.pm line 661.
set_test.pl: ERROR: died while trying to run COMMAND
launch_set.pl lambda --numcpus 1 --numnodes 20
set_test.pl: See if you have the correct software installed for this command.
6400 at /home/arahat/lyve-SET-1.1.4f/scripts/set_test.pl line 94.
make: *** [test] Error 25


I think the main problem is the "mpileup: unrecognized option --region", which seems to be blank. In the beginning of the block, it says creating pileup and then it immediately removes the file, probably to create the pipeline file.

I cannot change the code for Lyve-SET pipelining or create shell scripts for the pipeline because I am only configuring my server for use with the genome sequencing pipeline. Would this be a problem with the data set or some tool that I need to install? Any insight would be appreciated.

• What version of samtools mpileup to you have installed? What version of varscan are you using? Did you install the two tools separately or did varscan come with samtools? I have version 1.3.1 and there is a --region option for mpileup. Dec 27 '17 at 16:56
• Have you tried running samtools mpileup -f lambda/reference/reference.fasta -Q 0 -B --region NC_001416.1 /home/user/lyve-SET-1.1.4f/lambda/bam/NC001416.fasta.wgsim.fastq.gz-reference.sorted.bam directly to see if the command works outside the context of varscan? Dec 27 '17 at 16:59
• I installed samtools version 0.1.19 using yum install. Varscan was installed separately through make install from Lyve-SET. I did not try to run samtools mpileup separately because all the commands are implemented through Lyve-SET, which is how it will be run by the user. I will try reinstalling varscan and running the samtools mpileup command separately. Thank you for the help. Dec 27 '17 at 17:06
• I would suggest installing a more recent version of samtools. That version is from 2013 is likely is not compatible with the version of varscan you installed Dec 27 '17 at 17:15
• Yup. I noticed that yum patches but does not update the packages so I am manually installing the newest versions of all the tools specified in Lyve-SET. Thank you for bringing this to my attention. Hopefully it works after I reinstall. Dec 27 '17 at 17:21

The samtools v0.1.19 manual confirms that there is no --region option. In that version there is only the -r argument to pass in the targeted region. If you update to a newer version of samtools this should execute correctly