I just want to check my experiment design with you:
I was given RNA-SEQ samples from some genetically engineered bacteria. So those bacteria contain their basic genome and an insert (pET plasmid). Goal: quantify the expression of the plasmid.
What I've done:
Bowtie2: aligned the reads to the reference genome; extracted unaligned genes.
Trinity: created a transcriptome assembly using the unaligned reads (assuming they are the ones corresponding to the plasmid).
Trinity: Align reads [Bowtie2] and estimate abundance where I'm feeding it the transcriptome assembly from step 2 and the RNA-SEQ data.
Does it make sense?