Hello Galaxy Users-

I've been using the Main Galaxy server to work on an RNA-Seq project for a
non-model plant, and I've noticed that my output from Tophat and Cufflinks
might not be as good as I'd like.  I have a reference transcriptome
assembled in Trinity, and it is based on the same Illumina-generated 100 bp
reads I'm trying to map to it.  When I use Tophat to map the reads to the
reference transcriptome (I have trimmed the reads and filtered the lower
quality ones), only about 10% of the reads actually map, so I go from
30,000,000 reads before mapping to 3,000,000 that are actually mapped.
 Therefore, I feel like I'm losing a lot of data.  When I've changed the
parameters to allow for more mismatches, not many more reads seem to map,
and in many cases, the Tophat run fails and I receive the error
message: "*Settings:
Output files: "/tmp/
3030460.cyberstar.psu.edu/tmpWbxTnm/dataset_5530451.*.ebwt" Line rate: 6
(line is 64 bytes) Lines per side: 1 (side is 64 bytes) Offset rate: 5 (one
in 32) FTable chars: 10 Strings: unpacked Max bucket size: def"*.  I've had
similar numbers of reads map with Bowtie by itself and BWA as well.  I've
also tried mapping the reads to the assembled isoforms (contigs) of the
transcriptome, and this results in many more reads (close to 90%) being
mapped.  Therefore, I figure the reads should map to the reference
transcriptome, and I'm not sure why this isn't happening.

The other issue I've run into is that in Cuffdiff only about 4,800 genes
appear in the output files as being tested for differential expression.
 There are approximately 100,000 genes in the reference transcriptome, so I
was thinking that there should be more than ca. 4,800 that are tested for
differential expression.  Should each gene be tested?  Does Cuffdiff just
not report some of the genes that are not differentially expressed, or is
the program not testing all of the genes?

If anyone can provide some help, guidance, or a suggestion, I'd greatly
appreciate it.  Thanks, and take care.

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