Pacbio Rna-Seq Protocol

Question

[Botond Sipos]

Does anybody know about any paper/website describing the wetlab procedure(s) for RNA-seq using the PacBio platfrom?

Answer 1

[lexnederbragt]

The only source I know of is this poster: http://www.pacificbiosciences.com/pdf/Poster_Full_Length_cDNA_Sequencing_PacBioRS.pdf

[Botond Sipos]

Thanks! It's more than nothing...

Answer 2

[mchaisso]

I'm not sure about the sample preparation, but you can do alignment with GMAP. Contrary to popular belief, most (>95%) align properly without error correction with another platform. Fortunately, this is distributed across all genes (in the benchmarking I did) and so any isoform is usually detected if there are more than 3 or 4 reads that sample it.

[lexnederbragt]

I was going to suggest blasr, but something tells me you are the developer of this program :-) We really like blasr!

[mchaisso]

Yup, that's my code, glad you like it. As always, the more the feedback the better.

Blasr performs very poorly on spliced alignment. The introns violate some of the indel assumptions built into the code, and so you will often see part of a transcript mapped but many exons missing. I implemented some of the internals required to do spliced alignment in a blasr framework, but never finished it since it there were diminished returns relative to GMAP out of the box on every dataset I tested. If there is a dataset that defies mapping, let me know.

-mark