Question: Cuffdiff Fatal error: Matched on error
0
gravatar for rkp21
18 months ago by
rkp2120
rkp2120 wrote:

Hello,

I am doing my first RNA seq data analysis using galaxy. I have assembled my transcripts using Cufflinks and I merged my Cufflinks files using Cuffmerge. I am now trying to look at differential expression in the Cuffmerge .gtf output. I am trying to use Cuffdiff, but obtain the following error everytime I run the program:

Fatal error: Matched on error [14:34:45] Loading reference annotation. [14:34:53] Inspecting maps and determining fragment length distributions. BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute Processed 27666 loci.
Performed 315123 isoform-level transcription difference tests Performed 208521 tss-level transcription difference tests Performed 145119 gene-level transcription difference tests Performed 0 CDS-level transcription difference tests Performed 62122 splicing tests Performed 41198 promoter preference tests Performing 0 relative CDS output tests Writing isoform-level FPKM tracking Writing TSS group-level FPKM tracking Writing gene-level FPKM tracking Writing CDS-level FPKM tracking Writing isoform-level count tracking Writing TSS group-level count tracking Writing gene-level count tracking Writing CDS-level count tracking Writing isoform-level read group tracking Writing TSS group-level read group tracking Writing gene-level read group tracking Writing CDS-level read group tracking Writing read group info Writing run info

It looks like there is a problem while loading my reference annotation? I can't see why this would be a problem since I have used the same reference annotation for running cufflinks and cuffmerge. Let me know if there is a solution or if we need to update cuffdiff. We currently use cuffdiff v2.2.1.3 toolshed.g2.bx.psu.edu/repos/devteam/cuffdiff/cuffdiff/2.2.1.3

EDIT: I have tried running my analysis several times and decide to just start playing with the cuff diff output. I had 4 conditions and three replicates for each condition. My Cuff diff gene expression comparison file has 214,537 lines. My gene expression Cufflinks files have 38-40,000 lines. So my Cuff diff file has approximately the correct number of lines. Let me know if there is a flaw in my logic, but I think I have my data, just the CDS files failed to load, which caused the whole cufflinks program to give an error. I think only the CDS files were not generated.

ADD COMMENTlink modified 17 months ago by Jennifer Hillman Jackson23k • written 18 months ago by rkp2120
1
gravatar for Jennifer Hillman Jackson
17 months ago by
United States
Jennifer Hillman Jackson23k wrote:

Hello,

Where are you using Galaxy? Local, Cloud, public site (http://usegalaxy.org) or other? If http://usegalaxy.org, or this can be replicated there, a bug report can be sent in that will help in discovering what the exact problem is if the rest of the reply below does not uncover it. https://wiki.galaxyproject.org/Support#Reporting_tool_errors

That said, part of the error appears to be associated with the input mapped BAM datasets. Were these generated with Tophat or HISAT? The XS tag would be present if so. If not, try one of those tools. See the tool manual for how this tag is used (or see related questions to the right here >>> or you can even just google "Cuffdiff XS" to find much online discussion). http://cole-trapnell-lab.github.io/cufflinks/

There could also be a reference genome mismatch problem. And potentially an issue with the reference annotation. Try using the iGenomes GTF if available for the target genome (this sources contains all of the attributes Cuffdiff utilizes - many other sources do not). http://support.illumina.com/sequencing/sequencing_software/igenome.html https://wiki.galaxyproject.org/Support#Reference_genomes

Hopefully this helps you to find a solution, Jen, Galaxy team

ADD COMMENTlink written 17 months ago by Jennifer Hillman Jackson23k

Hi Jen,

Thanks for the reply.

I initially tried this on a local galaxy instance, but was able to replicate the problem on usegalaxy.org as well. That being said I did my alignment with HISAT2 on my local galaxy instance and brought the BAM files and the Cuffmerge from my local galaxy instance directly to usegalaxy.org.

It seems the "XS tag" is not a standard of BAM files, but is nececessary to run Cufflinks since cufflinks require the XS tag. I found an option in HISAT2 if I specify the alignment parameters I can specifiy to "report alignments specifically tailored for cufflinks". Hopefully this will solve the XS tag issue. I am currently downloading the reference annotation from iGenomes and will rerun my analysis using this reference annotation.

I will redo my alignment as specificed above and if I am still getting issues I will send a bug report.

Best, Ravi

ADD REPLYlink written 17 months ago by rkp2120

Hi Ravi, Thanks for following up. Posting back what works would be great! Jen

ADD REPLYlink written 17 months ago by Jennifer Hillman Jackson23k

Hi Jen,

If I specify "report alignments specifically tailored for cufflinks" I am able to remove the XS tag error. However I still haven't been able to successfully run Cufflinks. I'm actually running into a different error now which I describe here: Error while running cuffdiff "Fatal error: Exit Code 1()"

Best, Ravi

ADD REPLYlink written 17 months ago by rkp2120

Sorry you are having more problems. I just replied to that post. Details are in that thread. Jen

ADD REPLYlink written 17 months ago by Jennifer Hillman Jackson23k
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