Colleagues,
I am having trouble running cufflinks with an annotation file on the public Galaxy. The assembled transcripts gtf file has all the FPKM at 0 although the gene expression and transcript expression tab files have values for FPKM.
I have seen the SEQanswers threads about the compatibility of tophat bam files relative to chromosomes labeled as 1,2,3… versus Chr1, Chr2, Chr3… I am using the iGenomes bovine UMD3.1 genome and annotation file (chromosomes are 1,2,3) from the history. I altered the gtf file to Chr1, Chr2, Chr3… but it did not help.
Another potential discrepency/conflict is that the genome and gtf file have the bosTau6 database attribute from when I uploaded them. However I am running them from the history (bosTau6 is not an option for tophat). I do not seem to be able to remove the attribute.
Am I missing something else?
Here is the command line Info: cufflinks v1.0.3 cufflinks -q --no-update-check -I 50000 -F 0.050000 -j 0.050000 -p 8 -G /galaxy/main_database/files/003/142/dataset_3142240.dat -N -b ref.fa
Here is the details page Tool: Cufflinks Name: Cufflinks on data 69, data 4, and data 5: assembled transcripts Created: Nov 09, 2011 Filesize: 44.6 Mb Dbkey: bosTau6 Format: gtf Tool Version: Input Parameter Value SAM or BAM file of aligned RNA-Seq reads 4: Tophat for Illumina on data 4 and data 69: accepted_hits Max Intron Length 50000 Min Isoform Fraction 0.05 Pre MRNA Fraction 0.05 Perform quartile normalization Yes Conditional (reference_annotation) 1 Reference Annotation 5: iGen_UMD3_1_genes.gtf Conditional (bias_correction) 0 Conditional (seq_source) 1 Using reference file 69: UMD31_iGen_1-29X.fa Conditional (singlePaired) 0
Cordially, Chris