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Welcome to the Full-Length-RNA-Analysis-Best-Practice wiki!
As we know, PacBio Sequel SMRT cell contain about one million ZMWs. In generally, each ZMW product one effective transcript, theoretically, we can maximumly get one million transcripts in one Sequel SMRT cell, but in the real world, due to the loading efficiency and polymerase activity, we just can get about 0.2~0.3 million transcripts in one SMRT cell, this is the major limit for its widespread use in gene and isoform level quantification.
Here, we provide an innovated and patented library construction protocol, which can sequence multi-transcripts in one ZMW via linking RNA isoforms. Specifically, it can yield up to ~2.5 million full-length transcripts in one Sequel SMRT cell using V3.0 chemistry.
On Sequel II system, using V2.0 chemistry, we already can yield ~15 million high quality full-length transcripts in one SMRT cell. We will plan to release and publish this data in near future.This is an exciting results for PacBio IsoSeq users we believe.
As above figure shows, different from the official protocol, we using an unique experimental techniques to connect different transcripts molecules together to construction SMRTbell library for sequencing. After sequencing, we using specific bioinformatics tools to separate different transcripts molecules.
Above figure is an example we sequenced, this is an plant samples, we sequenced three times in this sample with difference library construction method. As the result shows that IsoLink-seq can yield ~2.6 million full-length transcripts in one SMRT cell with high gene and isoforms detection ratio comparable to NGS.
Above figure show that IsoLink-seq VS NGS have 0.923 correlation rate in gene quantification, it's significantly higher than official protocol.
Poly(A) length correlates with mRNA half-life. Widespread uridylation and guanylation at the downstream of poly(A) tail, implicate their generic roles in mRNA stability control. Full-length polyA tail is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.
(TAIL-seq: genome-wide determination of poly(A) tail length and 3' end modifications. Chang H, Lim J, Ha M, Kim VN. Mol Cell. 2014 Mar 20; 53(6):1044-52.)
PacBio official protocol use 27bp oligo dT primer. Theoretically, only ~30 polyA tail can be detected.
We developed a new protocol that can detect full-length polyA tail.
As above figure shows that PacBio official protocol just can detect median ~30bp polyA tail, in contrast, our new protocol able to detect longer polyA tail.