Hi I am a newbie in RNA seq any help will be deeply appreciated. i have 100 cases and 100 controls for each case and each control i have four different cell type and each cell type will be treated with two chemical (so 4x2x2 possible situation). I want to check the DE between cases and controls, between cell types and between treatment and I have pretty much no idea where to start. so far I came to conclusion that using kallisto is probably a good option for expression quantification and as it doesn't work with DESeq i thought maybe sleuth can be used for DE analysis but I'm not sure if I'm right. I'll be grateful if you can explain briefly how so many groups can be compared trustable.
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just to be clear kallisto+sleuth might not be the best option, i'm basically looking for some insight to know how I can compare all these different conditions get the DE between cases and controls for each cell type and under each treatmrent (they are healthy and diseased people)
Hi sam,
Did you try to adapt sleuth' introduction exercice (https://rawgit.com/pachterlab/sleuth/master/inst/doc/intro.html) your assay?
I will look to it thanks
Be aware that this tutorial lacks of the sleuth_wt() analysis you would run with your samples. It will provide you a wealth of information and data useful for you, as well as access to some functions in the live shiny application not available if you only run the likehood assay
You can combine kallisto with DESeq2, so you don't have to use sleuth. More about this can be found here: https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.pdf under tximport.
You'll find plenty of examples and questions about complicated designs for DESeq2 :p
this seem to do what I want! thanks a lot!