if one has project, with say treatment vs control samples (n = 3 x 2), and wants to understand which isoforms are differentially expressed, will IsoSeq give enough coverage? Is the trade-off cost / coverage / long-read enough to not do short-read?
The final goal of the analysis would be (1) understand splicing changes driven by the treatment, and (2) use some in qPCR in follow up experiments. Whilst long-reads will give undoubtedly a better picture of the transcriptome, that is, which isoforms are expressed in each sample, my concern is that the number of reads per isoform in any given sample will be too low to say if an isoform present in both groups is significantly more / less expressed in one of the groups with any confidence. It doesn't help that I can't find much in way of literature for this use case. Short read outputs enough coverage, but the results, specially for anything other the most simple cases of exon skipping are hard to inpterpret - thus why I am considering long-read.
- I was thinking of 2-3 flow cells for 6 samples
- not interested in an hybrid approach.