Isoform Sequencing: Unveiling the Complex Landscape in Eukaryotic Transcriptome on the PacBio® RS II

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths.  Here, we describe the Iso-Seq method for sequencing full-length cDNA libraries using long reads generated by the PacBio RS II.

To demonstrate our method for sequencing long and often rare isoforms, rat muscle RNA was prepared using the Clontech^® SMARTer^® cDNA preparation kit followed by size-selection with the BluePippin™ device from Sage Science to extract transcripts from 1-3 kb and 3-6 kb. We sequenced 77 SMRT^® Cells and obtained 4.2 million reads, of which ~1 million were putatively full-length as determined by the presence of 5’ and 3’ cDNA primers and polyA tail signal. Within the 3-6 kb fraction, 44% of the full-length reads were over 3 kb. We developed a novel error-correction method to differentiate reads between isoforms into separate clusters for consensus calling. This strategy generated full-length transcripts up to 4.5 kb with ≥ 99% post-correction accuracy. Compared with known rat genes, our method not only recovered many currently annotated isoforms, but also several unannotated isoforms with RefSeq homologs. Additionally, we detected alternative stop sites, extended UTRs, and retained introns. To conclude, the Iso-Seq method provides a new opportunity for researchers to study the complex eukaryotic transcriptome even in the absence of reference genomes or annotated transcripts.