Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells

Ashley Byrne,Mark Akeson,Hugh E Olsen,Charles Cole,Christopher Vollmers,Rebecca M Dubois,Theron Palmer,Miten Jain,Anna E Beaudin,E Camilla Forsberg

doi:10.1038/ncomms16027

Ashley Byrne, Mark Akeson + Show 8 more

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https://doi.org/10.1038/ncomms16027

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Abstract

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level.

Highlights

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level
The data we present here shows that RNAseq studies using the Oxford Nanopore Technologies MinION sequencer have the potential to redefine the level of information gathered by a single RNAseq experiment
By benchmarking our experimental and computational pipelines on Oxford Nanopore Technologies (ONT) MinION data derived from a mix of synthetic transcripts, we showed that our approach identifies the location of transcription start and end sites as well as splice sites in a genome

Summary

Introduction

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. We investigate whether RNAseq using the longread single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. It is important to determine the true transcriptional diversity of cells This requires that gene expression is analysed at the gene-level and at the isoform-level. Computational tools can be used to assemble full-length transcripts from these reads, but different assembly algorithms can result in conflicting outcomes and varying overall assembly quality[11] To offset this limitation of short-read RNAseq, studies have successfully used both single-molecule long-read PacBio Iso-Seq and synthetic long-read MOLECULO methodologies[12,13,14,15] to sequence full-length complementary DNA (cDNA). The Oxford Nanopore Technologies (ONT) MinION has been used to analyse full-length cDNA samples derived from both defined synthetic RNA molecules, as well as RNA from tissue culture cells[16,17]

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