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, researchers from the University of California–Santa Cruz 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 their experimental and computational approaches on a mixture of synthetic transcripts, they analyzed individual murine B1a cells using a new cellular indexing strategy. Using the Mandalorion analysis pipeline they developed, the researchers identified thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. They 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. These results show that not only can they identify complex isoforms, but also quantify their expression, at the single cell level.
Experimental Design and Analysis Pipeline
a) Schematic of experimental design. FACS-sorted single B1a cells were lysed. PolyA-RNA was then reverse transcribed and PCR amplified using template switching. Full-length cDNA was split into two reactions. Half of the reaction was tagmented by Tn5 and sequenced using a Illumina HiSeq2500 sequencer. The other half of the reaction was ligated to ONT adapters and sequenced on an ONT MinION sequencer. b) Schematic of the Mandalorion pipeline used to analyze the ONT 2D read data.