Have any of you used Galaxy (https://usegalaxy.org/) to analyze NGS RNAseq sequencing data to search for new splicing forms of RNA? I am looking for a pipeline for data analysis in Galaxy. Which programs are the best to use to find new splicing forms?
Hi Robert,
The steps you need for such an analysis are:
1. align your data with a splice-aware genome alignment tool (I prefer the STAR aligner)
2. check for splice junctions that are present in your data but not in other annotations
You can perform step 1 using Galaxy (there is "RNA STAR" and other such aligners). The second step however is a bit more custom and as such not present as program. I suppose you could try to compare the junctions file from your alignment to a genome annotation file that you can download from ENSEMBL/UCSC/NCBI. You could bedtools as a tool for this, but it will be pretty tedious.
If you only have a few junctions/genes that you are interested in, you could also look "manually".
If you have some programming knowledge, I advise you to use Whippet (https://github.com/timbitz/Whippet.jl) for this kind of analysis. Their documentation is pretty good and it has a feature for finding novel splice junctions.
Best regards
Alexander
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