RNA part II: Isoform Detection Q&A
Here you can find the responses from our guest speakers to questions that we did not have time to get to during the seminar.
1. What tools did you use for your analysis?
We used JuncBase and Flair
2. how did you processed the raw data? what software you used to correct the raw data?
Because I was not the one to do the computational part I will direct you to our preprint that has all the information
3. How did you correct the raw data for isoform analysis? Did you try the canu for correction?
Again I will direct to our preprint for all the analysis of the data. https://www.biorxiv.org/content/10.1101/2020.07.06.190330v1
1. How did you choose minimap2 for alignment?
There is broad community agreement that minimap2 is the best tool for aligning nanopore cDNA reads. I’m not aware of any other aligners that come close in terms of accuracy, speed or ease of use.
2. Do you process data first with PinFish before using FLAIR?
I used these two tools separately. First I used Pinfish to cluster the nanopore reads and form a consensus of the splice junctions, in order to benchmark the performance of ONT isoform sequencing alone (without use of Illumina reads). Then, in order to leverage the strengths of both long-read and short-read sequencing, I generated a hybrid transcriptome using the nanopore read structures with junctions corrected using the more accurate Illumina reads. So, if using FLAIR, you input the raw ONT reads directly, it is not necessary to run Pinfish first.
3. Are the new intergenic potential lncRNAs polyadenylated only? I wonder this because of the need of polyA for ONT data
We actually didn’t do polyA capture for this study. Instead, we did ribosome depletion and random hexamer priming for library preparation, followed by the ONT 1D DNA library prep kit (LSK109).