Hello,

Galaxy Main

1.) I am having trouble adding annotations to my Tophat and Cufflinks
tools.
I used the Mus.Musculus 9MM reference using the *built in index. *For the
Tophat mapping but no annotations were available in the output files.
I then tried converting the the Ref Genome from the UCSC to a SAM file
using Sam Tools. Tophat would not recognize this but Cufflinks did. The
Cufflinks output file did not have the annotation either.

Any thoughts on the proper way to add annotations?



2.) I am also trying to filter the single mapped reads from the multiple
mapped reads that resulted from Tophat. After converting the output file
from Tophat I used the filter tool in the Sam Tools choosing *0x100 map is
not primary. *Afterwards I tried to run Cufflinks on the filtered output
only to have it fail.


My ultimate goal is to look at RNA seq gene expression. I know that I have
to upload my files -> groom using FASTQ groomer -> download a reference
sequence from UCSC -> convert the reference genome file to a usable format
->Run Tophat for mapping using the groomed file and the converted reference
annotation -> Filter the single mapped reads -> Run cufflinks using the
filtered single mapped reads from Tophat.

>From here I will continue with some other statistical analysis but right
now I need to get this basic pipeline to work.


Thanks,
Kristen Roop
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