We have a trinity transcriptome assembly for 6 muscles types, 3 of which have triplicate data. We generated a supertranscript but now I’m reading conflicting things on how to approach the differential analysis portion of this experiment.
We have no reference genome so if we go with HISAT2 —> cufflinks —> Cuffdiff —> cummerbund wed of course use the supertranscript as the reference for which the reads align back to and go from there.
We also toyed with the idea of using DESEq/EdgeR
Then it was suggested to do hisat2–> stringtie —> ballgown but then someone says that those programs are good for genome assisted analyses while salmon/kallisto —> sleuth is transcript level analysis. I’ve looked into this type of pseudo alignment but at this point I just really don’t know which pipeline to use. Furthermore, some people think transcript level analysis is better than gene level analysis but it seems that gene level analysis is more reliable.