your pid must also be empty. I think: -s <seq_dir> Causes cuffcompare to look into for fasta files with the underlying genomic sequences (one file per contig) against which your reads were aligned for some optional classification functions. For example, Cufflinks transcripts consisting mostly of lower-case bases are classified as repeats. Note that <seq_dir> must contain one fasta file per reference chromosome, and each file must be named after the chromosome, and have a .fa or .fasta extension. might help --- On Thu, 7/14/11, Richard Mark White <whiter3@yahoo.com> wrote: From: Richard Mark White <whiter3@yahoo.com> Subject: [galaxy-user] rna-seq CDS, splicing and TSS fails To: galaxy-user@lists.bx.psu.edu Date: Thursday, July 14, 2011, 7:53 AM Hi all,   So I am using cuffdiff to find significant differences between two samples (no replicates).  The transcript and gene differential expression works and I get significant values.  However, consistently, the TSS, CDS, and splicing differences return with "1 line" and no data.  I have tried multiple different GTF transcriptome reference files  - UCSC refflat, ensembl, but still no luck.    Maybe I am missing something very basic - can anyone give me advice here? Richard -----Inline Attachment Follows----- ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org.  Please keep all replies on the list by using "reply all" in your mail client.  For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:   http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at:   http://lists.bx.psu.edu/