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Single cell long read sequencing and perturbation analysis reveals splicing heterogeneity and logic

Updated: Sep 29

Yuntian Fu:Graduate Group in Genomics and Computational Biology, University of Pennsylvania, Philadelphia, PA, USA Heonseok Kim, Susan M. Grimes, Billy T. Lau, Anuja Sathe,:Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

Alternative splicing is a ubiquitous regulatory mechanism of gene expression that allows generation of more than one unique mRNA species from a single gene. It contributes to cell differentiation and lineage determination, tissue-identity acquisition and maintenance, and organ development. While the functional roles and specificity of alternative splicing at tissue level have been widely studied, single cell level alternative splicing analysis remains unexplored. There are two main facets of single cell isoform analysis. On the one hand, different isoforms for the same gene could have very different functions. Cells with similar gene expression could have very different splicing patterns and play different functional roles. Thus, single cell isoform analysis could further reveal the heterogeneity of individual cells, especially for neurons and immune cells, which preserve extensive alternative splicing. On the other hand, distribution of isoforms within individual cells remains unclear. When an alternative splicing event is detected at bulk level, it could happen on cell population level, in which individual cells have binary outcomes in splicing: some cells always spliced in a particular cassette exon, and other cells never spliced in the exon. This can indicate further cellular heterogeneity. Isoforms could also coexist within individual cells at a similar ratio across the cell population. This indicates the functional importance of both isoforms. Related knowledge is still poor thus more transcriptome-wide studies are required to reveal the distribution of isoforms among and within single cells, which is important to understand the function roles and regulation mechanisms for alternative splicing. The scarcity of single cell alternative splicing analysis is mainly due to technical limitations. Common high-throughput single cell RNA sequencing methods couldn't sequence the full transcripts. Long reads sequencing could overcome this problem but it suffers from frequent sequencing errors, which make accurate isoform identification and quantification challenging. To overcome this difficulty and characterize cellular heterogeneity of alternative splicing, here we build a single cell alternative splicing analysis pipeline based on Nanopore long reads sequencing to reveal single cell alternative splicing heterogeneity and regulation mechanisms.

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