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Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome.

TitleMicrofluidic isoform sequencing shows widespread splicing coordination in the human transcriptome.
Publication TypeJournal Article
Year of Publication2018
AuthorsTilgner H, Jahanbani F, Gupta I, Collier P, Wei E, Rasmussen M, Snyder M
JournalGenome Res
Volume28
Issue2
Pagination231-242
Date Published2018 02
ISSN1549-5469
KeywordsAlternative Splicing, Computational Biology, Gene Expression Regulation, High-Throughput Nucleotide Sequencing, Humans, Microfluidics, Molecular Sequence Annotation, Protein Isoforms, RNA Splicing, Sequence Analysis, RNA, Transcriptome
Abstract

Understanding transcriptome complexity is crucial for understanding human biology and disease. Technologies such as Synthetic long-read RNA sequencing (SLR-RNA-seq) delivered 5 million isoforms and allowed assessing splicing coordination. Pacific Biosciences and Oxford Nanopore increase throughput also but require high input amounts or amplification. Our new droplet-based method, sparse isoform sequencing (spISO-seq), sequences 100k-200k partitions of 10-200 molecules at a time, enabling analysis of 10-100 million RNA molecules. SpISO-seq requires less than 1 ng of input cDNA, limiting or removing the need for prior amplification with its associated biases. Adjusting the number of reads devoted to each molecule reduces sequencing lanes and cost, with little loss in detection power. The increased number of molecules expands our understanding of isoform complexity. In addition to confirming our previously published cases of splicing coordination (e.g., ), the greater depth reveals many new cases, such as Coordination of internal exons is found to be extensive among protein coding genes: 23.5%-59.3% (95% confidence interval) of highly expressed genes with distant alternative exons exhibit coordination, showcasing the need for long-read transcriptomics. However, coordination is less frequent for noncoding sequences, suggesting a larger role of splicing coordination in shaping proteins. Groups of genes with coordination are involved in protein-protein interactions with each other, raising the possibility that coordination facilitates complex formation and/or function. We also find new splicing coordination types, involving initial and terminal exons. Our results provide a more comprehensive understanding of the human transcriptome and a general, cost-effective method to analyze it.

DOI10.1101/gr.230516.117
Alternate JournalGenome Res.
PubMed ID29196558
PubMed Central IDPMC5793787
Grant ListP50 HG007735 / HG / NHGRI NIH HHS / United States
R01 HL122887 / HL / NHLBI NIH HHS / United States
S10 OD020141 / OD / NIH HHS / United States