We have been doing 100bp paired end rna-sq in our lab for quite a long time. But recently the sequencing company quoted 150 bp paired-end reads from HiSeq4000 at fairly cheaper price than 100 bp paired-end reads, for the same 12G clean data. And we are aware of the benefits of longer reads in rna-seq. We have some precious samples from patients and we are studying splicing events (intron retention etc).
From experience and analysis point of view,
- 100 bp reads did a fair job in detecting splice junctions since most of the splice sites were within 20bp from exon boundaries and these reads cover them well.
- We also look for lincRNAs and depth was not a problem. But doing 150 bp might increase the horizontal coverage but it could also decrease vertical depth (maybe not drastically).
- Our collaborators have always worked with 100bp reads and even though they acknowledge the benefits of longer reads they still wants to stick with 100bp for reasons (since analysis pipelines are "optimised" for 100bp reads).
- Only major difference between HiSeq2k and 4k is the sequencing chemistry.
cheaper price and longer reads is of-course tempting.
Since we have limited amount of RNA and don't want to mess up, question is what else benefits does 150 bp has over 100bp and is it worth shifting?