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