> > Brand new galaxy user here. > > I ran an RNA-seq Illumina experiment in which I compare cells from wild > type animals and cells from animals that have a deletion in a splicing > factor. Now I have my data in fastq format and need to do analysis to > figure out which transcripts are changed and how (see below). Problem is, I > have no idea whatsoever what to do. > > Can someone be so kind and write down a basic outline of analysis to > follow? > > My understanding from what I've been reading online is that once you have > fastq files you > > 1. Use FASTQ Groomer to convert to Sanger format > > 2. Evaluate the quality with FASTQ Summary Stats (and get boxplot of data) > > 3. Trim reads if their quality doesn't look good > What is considered "ok" quality? A score above 20? Is that the > mean score or the absolute score? How do I trim based on score only those > reads that have a low q score? (can I?) > > 4. Map the reads > What mapping software do you reccomend? BWA or Bowtie? Or Tophat? > What next? > Let's say that anything after the trimming is very fuzzy. > > > The questions I am interested in are > - What transcripts are upregulated / downregulated in mutant vs control ? > (I have 3 replicates of each) > - Are there introns that are retained in mutant (but not or less in > control)? > - Are there exons that are excluded in mutant (basically, I want to at > patterns of alternative splicing..) > > > Sorry for the very long message, but I have no idea who else to ask. > > Thanks!! > > > > > > >
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