Aligning RNA-Seq reads to a reference genome using TopHat in GenePattern


Hello from GenomeSpace! In this video step, we will use the GenePattern tool to align RNA-Seq reads to a reference genome using the TopHat module. Launch GenePattern by clicking on the icon in the toolbar. Click on the Modules tab and search for the term ‘TopHat’. Click on the TopHat module when it appears in the window. You can also find modules by browsing through the entire listing, but searching is usually the most efficient method. We will run TopHat on a pre-constructed Bowtie index. Choose ‘Saccharomyces_cerevisiae_sacCer3_UCSC’ from the drop down menu. The reads pair one parameter requires our GenomeSpace file, WT.fastq.gz. Navigate to the file under the GenomeSpace tab, then click and drag the file to the module parameter. We will provide a GTF file by clicking on the file sacCer3_SGD.GTF, in our GenomeSpace sidebar. Choose ‘Send to GTF file’ to load it into the TopHat module. We don’t want to find novel junctions, so set this parameter to ‘No’. Finally, choose an output prefix such as ‘WT_readspair’. Click the ‘Run’ button. A spinning blue circle next to your job indicates that it is running. Now while our job is running, we will start up another TopHat job to align the reads from our mutant strain. To reuse a previous job as a template for a new one click on the job and then choose ‘Reload job’. Delete the old wild-type file by clicking on the ‘x’ on the right. Click and drag to load the SFP1KO.fastq file in its stead. Don’t forget to change the output prefix to something like ‘SFP1KO_readspair’ so that you can distinguish between the two job results. Click ‘Run’. TopHat can take several hours to run on the GenePattern public server depending on your place in the queue and the size of the files being aligned. Once the jobs finish, save the BAM files to GenomeSpace. This is easily done by clicking on the appropriate files under the completed job choosing “Save to GenomeSpace’ and selecting a directory. Remember to return to GenomeSpace and refresh the directory to view your files.

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