Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq

Cassandra Mcgill,Barbara J Wold,Diane Trout,Elisabeth Rebboah,Heidi Liang,Ali Mortazavi,Gabriela Balderrama-Gutierrez,Isaryhia Rodriguez,Katherine Williams,Fairlie Reese

doi:10.1186/s13059-021-02505-w

Cassandra Mcgill, Barbara J Wold + Show 8 more

Open Access

https://doi.org/10.1186/s13059-021-02505-w

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Abstract

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Highlights

Alternative transcript isoform expression is a major regulatory process in eukaryotes that includes differential transcription start sites (TSS) selection, RNA splicing, and TES selection
We found when comparing the ratio of spliced to unspliced reads in both read formats that there was typically a higher proportion of spliced reads detected in the short reads per cell versus the long reads, which may be due to the overall higher probability of sequencing an intronic region per read in long
Chromatin accessibility of myogenic marker genes distinguishes Myoghi and Pax7hi 72 h nuclei To assess chromatin accessibility in the groups of nuclei we identified with LR-Splitseq, we performed snATAC-seq on matching timepoints

Summary

Introduction

Alternative transcript isoform expression is a major regulatory process in eukaryotes that includes differential TSS (transcription start site) selection, RNA splicing, and TES (transcription end site) selection These differential choices sculpt the transcriptome and its resulting proteome during development, across cell types and in disease states. Long-read RNA-seq has been adapted to single-cell sequencing using high-throughput microfluidics-based methods [1,2,3,4] Some of these studies sequenced the same cells with both PacBio and Illumina technologies and relied on short-read gene quantification to cluster and characterize cell types, Rebboah et al Genome Biology (2021) 22:286 while using the long reads to identify full-length isoforms [2, 4]. These prior approaches used expensive equipment, such as microfluidics platforms, and/or applied very high amounts of long-read sequencing whose expense limits routine and extensive application

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