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.
