Hi Lizex, > I've started analyzing my RNA-Seq data for two time points: Day0 and Day4 for > control and treated. I've done aligning the data to the reference genome > using Tophat. I've removed duplicates from the data sets. Could somebody > please tell me, how important is it to remove duplicates and how will it > influence my results if I don't remove?
This depends on whether you are removing duplicates in your fastq data and/or multi-mapping reads either using Tophat or post-processing steps. In any case, this approach that will affect quantitation outputs from Cufflinks and likely transcript assemblies as well. > I want to start with Cufflinks all the way through to Cuffdiff. Where do I > start since there are just so many options (in the manual) to choose from? > What do I look for? > Here's a tutorial that will help you get started with RNA-seq analysis: http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise Galaxy makes it easy to experiment with different parameter values, so you'll want to read the Cufflinks/compare/diff manual and adjust parameters that are relevant to your data: http://cufflinks.cbcb.umd.edu/manual.html In general, RNA-seq studies look at (a) transcripts assembled; (b) expression values; and (c) differential expression estimates. Good luck, J.
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