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  • paulieme60 paulieme60 Mar 19, 2014 4:31 PM Flag

    Proceedings of the National Academy of Sciences

    Wednesday, March 19, 2014 PacBio Blog--- Assessment of Highly Complex Alternative Splicing of Neurexins Performed with SMRT Sequencing
    A new paper in the Proceedings of the National Academy of Sciences from the laboratories of Stephen R. Quake and Thomas C. Südhof (both at Stanford University) describes the direct, full-length transcript sequencing of RNA molecules that are essential to synapse formation in the mammalian brain. The team used Single Molecule, Real-Time (SMRT®) Sequencing to analyze full-length mRNAs from different members of the neurexin gene family and used that information to examine alternative splicing events.
    For this study, researchers used the PacBio® platform to sequence transcripts generated by three neurexin genes in adult mice. “Read lengths of up to 30 kb enabled us to identify all of the splice combinations within a single transcript,” they report. With sequencing reads representing more than 25,000 full-length mRNAs, the team made several important discoveries. These include: a novel alternatively spliced exon; even higher isoform diversity than was anticipated; and the finding that splicing events seem to occur independently of one another. The team was able to map out the full transcript landscape for a neurexin gene, showing alternative splicing at all six canonical sites as well as at several noncanonical sites.
    Being able to directly assess alternative splicing not only provided evidence for suspected isoform diversity, but also revealed “that neurexins are likely even more polymorphic than previously thought,” the team reports. Based on their observations from SMRT Sequencing, they calculated how many neurexin variants were possible in total. “We observed in this manner a minimal diversity of 1,159 isoforms for Nrxn1a, 1,120 isoforms for Nrxn3a, and a total of 152 isoforms for all three ß-neurexins,” they write. “Thus, earlier estimates of 2,000–3,000 neurexin (For full story go to PACB website,click on blogs).

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