Hello,<div><br></div><div>Galaxy Main</div><div><br></div><div>1.) I am having trouble adding annotations to my Tophat and Cufflinks tools. </div><div>I used the Mus.Musculus 9MM reference using the <i style="font-weight:bold">built in index. </i>For the Tophat mapping but no annotations were available in the output files.</div>
<div>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.</div><div><br></div>
<div>Any thoughts on the proper way to add annotations?</div><div><br></div><div><br></div><div><br></div><div>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 <i style="font-weight:bold">0x100 map is not primary. </i>Afterwards I tried to run Cufflinks on the filtered output only to have it fail.</div>
<div><br></div><div><br></div><div>My ultimate goal is to look at RNA seq gene expression. I know that I have to upload my files -&gt; groom using FASTQ groomer -&gt; download a reference sequence from UCSC -&gt; convert the reference genome file to a usable format -&gt;Run Tophat for mapping using the groomed file and the converted reference annotation -&gt; Filter the single mapped reads -&gt; Run cufflinks using the filtered single mapped reads from Tophat.</div>
<div><br></div><div>From here I will continue with some other statistical analysis but right now I need to get this basic pipeline to work.</div><div><br></div><div><br></div><div>Thanks,</div><div>Kristen Roop</div>