Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events

Abstract

Alternative splicing shapes mammalian transcriptomes, with many RNA molecules undergoing multiple distant alternative splicing events. Comprehensive transcriptome analysis, including analysis of exon co-association in the same molecule, requires deep, long-read sequencing. Here we introduce an RNA sequencing method, synthetic long-read RNA sequencing (SLR-RNA-seq), in which small pools (≤1,000 molecules/pool, ≤1 molecule/gene for most genes) of full-length cDNAs are amplified, fragmented and short-read-sequenced. We demonstrate that these RNA sequences reconstructed from the short reads from each of the pools are mostly close to full length and contain few insertion and deletion errors. We report many previously undescribed isoforms (human brain: ∼13,800 affected genes, 14.5% of molecules; mouse brain ∼8,600 genes, 18% of molecules) and up to 165 human distant molecularly associated exon pairs (dMAPs) and distant molecularly and mutually exclusive pairs (dMEPs). Of 16 associated pairs detected in the mouse brain, 9 are conserved in human. Our results indicate conserved mechanisms that can produce distant but phased features on transcript and proteome isoforms.

Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome

“…6A). This reduced presence of constitutive exons in lncRNAs is consistent with increased isoform diversity through skipping of exons previously considered constitutive (Sharon et al 2013;Tilgner et al 2013Tilgner et al , 2014Tilgner et al , 2015 and universal alternative splicing (Deveson et al 2017) in lincRNAs. This procedure performs one test per gene, and we correct for multiple testing using the Benjamini-Hochberg method (Benjamini and Hochberg 1995).…”

“…However, a mismapped read giving a false-positive junction for a droplet can also lead to a false-positive collision. It is expected that, for highly expressed genes, it is more likely to have two molecules in a droplet (see Tilgner et al 2015 for calculations). In our spISO-seq data, most spliced genes were detected in no more than 2000 (out of >200,000) droplets (Fig.…”

“…Earlier genome-wide work showed correlated inclusion patterns across tissues (Fagnani et al 2007), but distinct isoform arrangements can underlie this observation. Yet, despite very important insights (Helfman et al 1986;Cramer et al 1997;Fededa et al 2005;Fagnani et al 2007;Tilgner et al 2015), the understanding of exonic variability in full-length molecules is far from complete, because of the experimental difficulties of monitoring multiple variable sites in a single long molecule. Long-read RNA sequencing allowed the interrogation of splice sites en masse along the molecule (Tilgner et al 2014.…”

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

“…Currently, there are three different long-read transcriptome sequencing platforms: Pacific Biosciences (PacBio) (Sharon et al 2013;Tilgner et al 2014;Li et al 2016), Moleculo (Tilgner et al 2015), and Nanopore (Oikonomopoulos et al 2016). Here, we have used the popular PacBio Iso-Seq protocol, which consists of full-length cDNA enrichment using the Clontech SMARTer kit followed by building single-molecule SMRTbell libraries with specific PacBio linkers that are subsequently sequenced.…”

“…All PacBio transcriptome papers discover thousands of new transcripts, propose classification schemes by comparing to a reference annotation, and find that the majority of novel transcripts appear in known genes (Au et al 2013;Sharon et al 2013;Tilgner et al 2015;Abdel-Ghany et al 2016;Wang et al 2016). However, details on the number, quality, and characteristics of these new calls can vary greatly.…”