I am trying to design an experiment to do differential experiment analysis on two breeds of one species and compare them. I have been suggested to do alternative splicing analysis and look for different variants using RNA-seq as well. The genome of the animal I am working with has been assembled, and I have access to its gff file. Do I need to do alternative splicing analysis in addition to differential expression as well? Also, please recommend software to do alternative splicing analysis for RNA-seq.
1 answer
it all depends on your first biological question. AS is an essential component of gene expression regulation so if you compare two breads of one specie I find relevant to study it in parallel of differential gene expression.
I suggest you, for AS analysis, to use STAR alignement tool in 2pass-mapping mode (to detect more splicing junctions) : https://physiology.med.cornell.edu/faculty/skrabanek/lab/angsd/lecture_notes/STARmanual.pdf
and for the statistical part, rMATS (http://rnaseq-mats.sourceforge.net/), that focus on compare the splicing junction between two conditions
PS : I studied AS two years ago so maybe better tools have been developed since
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