Question: galaxy biomina RSeQC bam/sam mapping stats error
0
gravatar for q568lvdk41089dje
3.2 years ago by
q568lvdk41089dje0 wrote:

I am trying to run the Galaxy Biomina RseQC BAM/SAM mapping quality stats

I have selected my uploaded bam file (generated using Tophat on the main Galaxy instance to map the BodyMap brain tissue paired end fastq files, provided in the shared data).

To make a smaller file for trial analysis, I am using only the reads that map to chr22

(ie. bam>sam> filter for c3=='chr22' and sam>bam)

I have selected 50 for the mapping quality minimum for unique reads (which I believe is the case for Tophat)

BUT

I get the following in information> stderror

Fatal error: Exit code 127 (An error occured during execution, see stderr and stdout for more information)

I get blank with stdout

Can anyone suggest what the problem might be?

Doiremac

rseqc biomina • 1.2k views
ADD COMMENTlink modified 3.2 years ago by Jennifer Hillman Jackson25k • written 3.2 years ago by q568lvdk41089dje0
0
gravatar for Jennifer Hillman Jackson
3.2 years ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

I cannot reproduce this error on a local or Cloudman Galaxy with a Tophat2 BAM dataset and the RSeQC tools installed from the Tool Shed using Galaxy version 15.07

To troubleshoot, first make certain that the tools and Galaxy version are up to date. Next, double check the format of the inputs - this is a very common reason for tool errors. Do other tool operations on the BAM dataset succeed (Samtools, Picard)? Other tools from this suite succeed? 

Please let us know how this goes, Jen, Galaxy team

ADD COMMENTlink written 3.2 years ago by Jennifer Hillman Jackson25k

OK thanks Jen I'll try and give you as much detail as pos

Using the main galaxy website usegalaxy.org (not sure of version but carried out on 6th August 2015 if that helps)......

I imported the (shared data) Body Map brain paired end fastq files. I then mapped them using Tophat v2.014 (the one available on usegalaxy.org) using the following settings: PE built-in Hg19 readgroup ID brain_readgroup LB brain_library PL ILLUMINA SM human_brain_tissue and other parameters at default

In usegalaxy.org I converted bam>sam (samtools galaxy tool v2.0) filtered for 'c3==chr22' (using filter galaxy tool v1.1.0) and back to bam again (sam>bam)

I then downloaded the bam and bai file locally (brain_chr22.bam and brain_chr22,bai) and uploaded the bam file to the http://biominavm-galaxy.biomina.be/galaxy/ website.

Within that, yes the bam file is successfully usable in their samtools (bam>sam), bedtools (create bedgraph of genome coverage) and in picard (mark dups and library complexity).

The problem seems to be with their RSeQC (v2.3.9) functions and my bam file.

I have tried 1. bam/sam mapping stats (with my bam file and also with the bam converted to a sam with their samtools converter) Failed with: stderr = Fatal error: Exit code 127 (An error occured during execution, see stderr and stdout for more information) stdout = blank

I have tried 2. read distribution (with my bam and a bed file generated from: ucsc table browser hg19, refgene, name2 = gene symbols for chr22 and with the txt file converted to a bed file using their tool "get data>generate bed file") Failed with: stderr = Fatal error: Exit code 127 (An error occured during execution, see stderr and stdout for more information)

/var/spool/torque/mom_priv/jobs/2151452.biominavm-ldap-pbs.biomina.be.SC: 1: read_distribution.py: not found

stdout = blank

I have tried 3. gene body coverage with the above files

Failed with:

stderr = Fatal error: Exit code 127 (An error occured during execution, see stderr and stdout for more information)

/var/spool/torque/mom_priv/jobs/2151464.biominavm-ldap-pbs.biomina.be.SC: 1: geneBody_coverage.py: not found

stout = blank

???

Your thoughts would be much appreciated, thanks in advance

Roisin

ADD REPLYlink written 3.2 years ago by q568lvdk41089dje0

Hi, 

From the description, this is almost certainly a problem on the other public Galaxy server at http://biominavm-galaxy.biomina.be/galaxy. Their contact information can be found here: https://wiki.galaxyproject.org/PublicGalaxyServers#Biomina

That said, the correct usage for filtering syntax is: c3== 'chr22'

Please note the way that quotes are used, as this differs from what you entered above, and I am wondering if the result of the filter produces a SAM/BAM dataset that actually contains more than headers. This can be manually examined when the data is in SAM format - or a "Group" function could work as well (omit lines that start with an "@" symbol, an option on this tool form). Or, various tools in the group SAMtools and Picard can help summarize the contents a BAM dataset. I know that you used other tools that appeared to execute successfully, but am not sure of the output contents. Tools will not necessarily "fail" (turn red) if there are no data lines and only headers in the dataset. I would be somewhat surprised if Bedtools returned a viable bedGraph dataset without mapping data line content in the input dataset, but this all seemed worth mentioning, just in case.

Hopefully this leads to a solution! Jen, Galaxy team

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by Jennifer Hillman Jackson25k

Hi Jen sorry for typo, but I did filter correctly c3=='chr22' I also did get viable output files from Picard, Bedtools, etc. I'll try and contact Biomina Thanks

ADD REPLYlink written 3.2 years ago by q568lvdk41089dje0
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