Hi, in my experiment design, I have RNA-seq data of three different tissues, and I want to take a look at the expression pattern of three tissues. But I have a question: how to generate a reference for my experiment design? Should I put the reads of three tissues together to get a assembly or generate three assemblies according to different tissues using Trinity? Thank you.
If you have the reference genome available for the organism from which the tissues were extracted , you can go for a what is known as a "reference" based transcriptome assembly. If you don't have a reference available as in case of certain non-model / less studied/ less known / less popular organisms, you can perform what is known as a "denovo master" assembly using all the 3 samples together.
Extra piece of information
Since a genome contains the sum of all introns and exons that may be present in a transcript, spliced variants that do not align continuously along the genome may be discounted as actual protein isoforms. Even if a reference genome is available, de novo assembly should be performed, as it can recover transcripts that are transcribed from segments of the genome that are missing from the genome assembly