I am currently following the tophat->cufflinks->cuffmerge->cuffdiff pipeline. Everything up until cuffmerge worked out well. I have been trying to run cuffdiff between 4 conditions x 3 replicates/condition, using the the cuffmerge output as the input gtf. After having done so, all cuffdiff output files, including the sqlite file, showed up as empty. I have previously performed the same analysis with the same parameters and annotation file and it went perfectly well. Not sure where the problem lies, since the annotation file contains all the attributes needed (i.e. gene_id, gene_name, p_id, transcript_id, tss_id. Example:
Seqname Source Feature Start End Score Strand Frame Attributes chr1 unknown exon 3214482 3216968 . - . gene_id "Xkr4"; gene_name "Xkr4"; p_id "P15119"; transcript_id "NM_001011874"; tss_id "TSS26513";
I am trying to re-run cuffdiff, but now under "Perform Bias Correction" -> "Reference Sequence Data" -> "Using Reference Genome" the drop down menu is absent and says "No options available."
Is anyone else experiencing the same problem or have a solution?
Cuffdiff worked when run without biased correction. There seems to be a correlation between empty cuffdiff output files and using the "bias correction" option. Any ideas?