Nanopore native RNA sequencing of a human poly(A) transcriptome

Abstract

High throughput cDNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions, to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications, and transcript haplotypes.

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…It is noteworthy, that a minimum aligned length of about 100 nt appears in the data sets (see Supplementary Fig. 3d) when using the native RNA sequencing protocol and minimap2 as mapping tool 16,40,56 . Unaligned reads had a median read length of 191 nt, in contrast to 572 nt for aligned reads (all data sets combined) suggesting that short reads could not be aligned properly.…”

“…The key steps of the library preparation are shown in Fig. 1a: after enzymatic polyadenylation, the RNA is reverse transcribed to increase sequencing throughput (recommended by ONT) 16 . Subsequently, a motor protein is added that feeds the RNA into the Nanopore.…”

“…methylations 14,15 ). As ONT-based native RNA-seq holds the capacity to sequence full-length transcripts, to identify RNA base modifications and to detect molecular heterogeneity in a transcriptome, the technology found widespread attention 16 . Recently, the technology was exploited to sequence viral RNA genomes 17–20 to gain insights into viral and eukaryotic transcriptomes 19,21–23 and to detect RNA isoforms in eukaryotes 24,25 .…”

Expression changes confirm genomic variants predicted to result in allele-specific, alternative mRNA splicing

“…Our protocol identifies low or non-penetrant allele-specific alternative splicing events through bioinformatic analysis, and either q-RT-PCR, exon microarrays or RNAseq data analysis. Allele-specific splicing can also be determined by full-length alternative isoform analysis of RNA (or FLAIR [Workman et al 2019]). Differentiated splice forms are associated with specific alleles in heterozygotes with exonic SNPs.…”

Nanopore sequencing reveals U2AF1 S34F-associated full-length isoforms

“…Recent studies have shown the utility of long-read approaches in capturing full-length mRNA isoforms, by constructing isoforms missed by short-read assembly methods (Oikonomopoulos et al, 2016;Byrne et al, 2017;de Jong et al, 2017;Tang et al, 2018;Workman et al, 2019). Moreover, long-read approaches have already been conducted using RNA derived from primary tumor samples harboring SF3B1 mutations, demonstrating its effectiveness in capturing mutant splicing factor transcriptome alterations (Tang et al, 2018).…”