cross species RNA-seq/orthologs

[KateShepard]

Dear Forum,

I am completely new to RNA-seq analysis so would be very appreciative of any advice regarding my query.

I have multiple RNA-seq samples from two closely related species, and would like to identify differentially expressed orthologous genes.

Is a correct approach to first identify orthologs between the two species using OrthoMCL, annotate those orthologs using (?), align all RNA-seq samples to the orthologs using bowtie2/tophat2, and assemble transcripts and identify differentially expressed transcripts using cufflinks and cuffdiff?

Thank you for your help and advice!
Kate

[colindaven]

Do you have a genome ? Where do you get your sequences from if not, transcript de novo assemblies ?

I would suggest proteinortho instead of orthomcl, as it is simpler in my experience.

I would use STAR instead of tophat2 if possible, featureCounts for read counting and edgeR/DEseq2 for DE analysis if it all works out.