July 11, 2016  |  General

First Comprehensive View of Alternative Splicing in Sorghum Powered by SMRT Sequencing

A paper from scientists at Colorado State University and the National Center for Genome Resources provides an in-depth view of the transcriptome of sorghum, a crop that’s important for human and animal food and also shows potential as a biofuel. Through this project, the team produced a new isoform analysis pipeline for community use and identified novel genes, as well as far more alternative splicing than had been expected for this plant.
The publication, “A survey of the sorghum transcriptome using single-molecule long reads,” comes from lead author Salah Abdel-Ghany, senior author Anireddy Reddy, and collaborators. The researchers were particularly interested in alternative splicing and alternative polyadenylation, two mechanisms that increase transcript diversity and may help plants adapt to stress. “Despite the fact that several large-scale RNA-seq studies have been performed in plants to analyse [alternative splicing], currently it is not known how many distinct splice isoforms are produced,” the team writes. “This is primarily due to challenges associated with short-read sequencing in accurately reconstructing full-length splice variants.”
To directly observe full-length splice isoforms, they turned to long-read SMRT Sequencing to characterize the transcriptome of sorghum seedlings. The scientists also developed the Transcriptome Analysis Pipeline for Isoform Sequencing, or TAPIS, to identify alternative splicing events and evidence of alternative polyadenylation. “The analysis of sorghum Iso-Seq data uncovered over 7,000 novel [alternative splicing] events, ~11,000 novel splice isoforms, over 2,100 novel genes and several thousand transcripts that differ in 3′ untranslated regions due to [alternative polyadenylation],” the team reports, noting that many of the novel genes are putative long non-coding transcripts. The total number of unique transcripts was nearly 28,000, covering more than 14,000 genes.
The scientists discovered a significantly higher rate of alternative splicing than had been expected for sorghum. “Previously, it was reported that pre-mRNAs of ~1,500 genes undergo [alternative splicing] in sorghum,” they note. “In this work, we demonstrate that this number is much higher.” Indeed, they found more than 10,000 alternative splicing events, compared with fewer than 3,000 included in existing gene models. The scientists performed a number of validation studies to confirm these and other novel findings and have made their TAPIS tool available for use in other organisms.
“Pacific Biosciences single-molecule long reads obtained using the Iso-Seq protocol offer a considerable advantage in transcriptome-wide identification of full-length splice isoforms and other forms of post-transcriptional regulatory events such as [alternative polyadenylation],” the team writes.

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