Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells

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, while the current standard for gene expression quantification, is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigated whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencing technology (ONT RNAseq) would be able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After successfully benchmarking our experimental and computational approaches on a mixture of synthetic transcripts, we analyzed individual murine B1a cells using a new cellular indexing strategy. Using the Mandalorion analysis pipeline we developed, we identified thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identified hundreds of genes expressed across B1a cells that displayed multiple complex isoforms, including several B cell specific surface receptors and the antibody heavy chain (IGH) locus. Our results show that not only can we identify complex isoforms, but also quantify their expression, at the single cell level.

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

“…To identify transcript isoforms, we used the R2C2 reads as input into a revised version of the Mandalorion tool (v3, github) 10 . We detected 21,358 transcript isoforms expressed from 9,971 gene loci.…”

“…However, evidence is accumulating that these epitopes might be absent in some transcript isoforms of these genes. Detailed analysis of this absence and possible consequences is hampered by the inability of RNA-seq to resolve transcript isoforms 10,15,16 . Determining isoform level transcriptomes of healthy and cancerous immune cells might therefore inform treatment decisions and future development.…”

Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain

“…To address these unknowns, we developed a straightforward and cost-effective approach using a long-read sequencing technology [32][33][34] . We combined long-range RT-PCR with long-read nanopore sequencing and a novel bioinformatics pipeline to characterise full-length CACNA1C coding sequences (CDSs) in post-mortem human brain.…”

AIDE: annotation-assisted isoform discovery with high precision

“…The non-parametric approach to learning the read generating mechanism makes AIDE a data-driven method and does not depend on specific assumptions of the RNA-seq experiment protocols. Therefore, a natural extension of AIDE is to combine the short but more accurate reads from the second-generation technologies with the longer but more error-prone reads generated by new sequencing technologies such as PacBio (Rhoads and Au 2015) and Nanopore (Byrne et al 2017). Jointly modeling of the two types of reads using the AIDE method has the potential to greatly improve the overall accuracy of isoform detection (Fu et al 2018), since AIDE is shown to have better precision than existing methods, and longer RNA-seq reads capture more splicing junctions and can further improve the recall rate of AIDE.…”