Galaxy-filter pileup error

[rudi283]

Hi,
The aim of my work now is to find SNPs in my dataset from Illumina GAII ( paired end) but when tried to filter pileup got this:

16: Filter pileup on data 14
An error occurred running this job: Use of uninitialized value in subroutine entry at /galaxy/home/g2main/galaxy_main/tools/samtools/pileup_parser.pl line 40, <IN> line 1.
Use of uninitialized value in numeric lt (<) at /galaxy/home/g2main/galaxy_main/tools/samtools/pileup_parser.pl line 41

I'm new Galaxy user, working with this kind of data for the first time...
I just had a first go and got an error... Could you please direct me on a right way of doing it as I must did something totally wrong
What I was doing:
-uploaded data in fastq format
-FASTQ groomer (Illumina 1.3+)
-FASTQ summary statistics
-FASTQ splitter
-Map with Bowtie for Illumina (paired end, typed my insert size)
-Filter SAM (flag1-read is paired YES; flag2-read is mapped in a proper pair-YES; flag3-read is umpapped-NO)
-SAM to BAM
-Generate pileup from BAM dataset
-Filter pileup

(not sure if I should have trimmed the reads first...)

I probably have been doing this all wrong from the beginning, so if anyone could tell me, what should I do to get the right thing would be great

[tnabtaf]

I'd suggest posting this question to the Galaxy-User list (http://lists.bx.psu.edu/listinfo/galaxy-user). You may get a good response there.

Before you do that, this information may help.

The 2 lines of code that are choking are:

40 die "Coverage column" . ($cvrg_column+1) . " contains non-numeric values. Check your input parameters as well as format of input dataset." if ( not isdigit $fields[ $cvrg_column ] );
41 next if $fields[ $cvrg_column ] < $cvrg_cutoff;

The three variables are set at:

13 my $cvrg_column = $ARGV[4]-1; # 1 based

15 my $cvrg_cutoff = $ARGV[6]; # unsigned integer

37 my @fields = split /\t/;

The coverage cutoff is set from the Galaxy interface. Check what value you are giving it.

If that's not the problem then there is a problem in how the coverage column number is being set (which I think is set by Galaxy), or some of the columns are missing.

When you post to Galaxy-User, please provide a few lines of the file that is being given to the filter pileup step.

[rudi283]

Thank you for the reply!
I'm trying to do the same for my other data as maybe there is something wrong with the data I uploaded to Galaxy. If it will be the same I'm going to do what you suggested. Thanks for help!

[rudi283]

This time is not good as well I don't have an error but the file is empty...
Looks like it's because when I try to generate pileup I get 3 columns with '0' values...

I'm not sure why Am I doing something wrong or there must be a problem with my fastq file?
Please help