Hi Kelly. I just wanted to follow-up on this issue. I did some more tests and it seems that the problem may be because the fragement headers in my custom reference multi-fasta contain the coordinates of the fragment in the form chr:start-stop. Upon mapping with bowtie, the resultant SAM file contains the headers (as expected). But the BAM-to-pileup tool does not play well with such headers. It seems the BAM-to-pileup tool expects headers in only chr format. Since there does not appear to be a tool on galaxy to convert chr:start-stop to chr format in a SAM file (note: this also requires modification of the next column in the SAM file), the solution at this point is to download the SAM file, locally apply the fix, re-upload the SAM file and run the BAM-to-pileup tool with a built-in index (mm9 in my case). Even at this step, I still can't use my custom reference for the pileup because it contains headers in chr:start-stop format. Thoughts?