Hi Noa, There are two issues: Using the correct genome and setting up paired data properly. 1 - Custom genomes Perhaps you have discovered this already, but to set the TopHat tool form to use a custom reference genome from the history, this option: "Will you select a reference genome from your history or use a built-in index?:" Should be set to: "Use one from the history" The form will then refresh, offering a new pull-down menu under the option: "Select the reference genome:" that now includes datasets from the right history panel (instead of genome names, native to Galaxy). Select the fasta formatted custom reference genome and initiate the job. I am not sure how you were able to get 100% mapped if the wrong genome was used ..., perhaps the genome you ended up mapping to was similar to the one you wanted to map to (different releases of the same species)? Or maybe you did use the genome from the history after all? Moving on to Cufflinks might give you some more information about the map quality (especially if used with a reference gene GTF file). 2 - Mated pairs Flagstat is not the best tool for the latest RNA-seq data. Instead, try "NGS: Picard (beta) -> SAM/BAM Alignment Summary Metrics or BAM Index Statistics". Hopefully this has been worked out or this is helpful! Apologies for the delay in reply, this question was overlooked during the holiday shuffle, Best, Jen Galaxy team On 12/13/11 1:17 PM, Noa Sher wrote:

I am trying to run Tophat on some Illumina data from an organism with no UCSC-supported genome. I have taken my Illumina data and run it through the FASTQ groomer. I input this data into tophat, and choose the built-in index option for ------------this was the problem --------------^^^^^^^^^^^^^^^-------- the reference genome, using an uploaded GenBank FASTA genome. I run this, but when I check the output by running *NGS: SAM Tools > flagstat* on the bam output from tophat, I get 0% properly paired (see below for the exact output). I have BLASTed the Illumina data just to double-check that I have not mixed files; it is in fact from the organism I am tophat-ing against. I would greatly appreciate input on what I may be doing wrong? Is the "built-in index" supposed to be just the genome sequence in FASTA format or is it something more complex? Thanks Noa

6667700 in total 0 QC failure 0 duplicates 6667700 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)

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