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

Nat Commun. 2017 Jul 19:8:16027. doi: 10.1038/ncomms16027.

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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / metabolism*
  • Benchmarking
  • Gene Expression Profiling / methods*
  • Mice, Inbred C57BL
  • Protein Isoforms / metabolism
  • Receptors, Cell Surface / metabolism*
  • Sequence Analysis, DNA
  • Sequence Analysis, RNA
  • Single-Cell Analysis / methods*
  • Transcriptome

Substances

  • Protein Isoforms
  • Receptors, Cell Surface