Dataset Information

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

ABSTRACT: A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

SUBMITTER: Tian L

PROVIDER: S-EPMC8582192 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

Tian Luyi L Jabbari Jafar S JS Thijssen Rachel R Gouil Quentin Q Amarasinghe Shanika L SL Voogd Oliver O Kariyawasam Hasaru H Du Mei R M MRM Schuster Jakob J Wang Changqing C Su Shian S Dong Xueyi X Law Charity W CW Lucattini Alexis A Prawer Yair David Joseph YDJ Collar-Fernández Coralina C Chung Jin D JD Naim Timur T Chan Audrey A Ly Chi Hai CH Lynch Gordon S GS Ryall James G JG Anttila Casey J A CJA Peng Hongke H Anderson Mary Ann MA Flensburg Christoffer C Majewski Ian I Roberts Andrew W AW Huang David C S DCS Clark Michael B MB Ritchie Matthew E ME

Genome biology 20211111 1

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. A ...[more]

PMID: 34763716

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Project description:BackgroundPosttranscriptional processing of precursor mRNAs contributes to transcriptome and protein diversity and gene regulatory mechanisms in eukaryotes. However, this posttranscriptional mechanism has not been studied in the marine macroalgae Gracilariopsis lemaneiformis, which is the most cultivated red seaweed species in China.ResultsIn the present study, third-generation sequencing (Pacific Biosciences single-molecule real-time long-read sequencing, SMRT-Seq) was used to sequence the full-length transcriptome of G. lemaneiformis to identify alternatively spliced transcripts and alternative polyadenylation (APA) sites in this species. RNAs were isolated from G. lemaneiformis under various treatments including abiotic stresses and exogenous phytohormones, and then equally pooled for SMRT-Seq. In summary, 346,544 full-length nonchimeric reads were generated, from which 13,630 unique full-length transcripts were obtained in G. lemaneiformis. Compared with the known splicing events in the gene models, more than 3000 new alternative splicing (AS) events were identified in the SMRT-Seq reads. Additionally, 810 genes were found to have poly (A) sites and 91 microRNAs (miRNAs), 961 long noncoding RNAs and 1721 novel genes were identified in G. lemaneiformis. Moreover, validation experiments showed that abiotic stresses and phytohormones could induce some specific AS events, especially intron retain isoforms, cause some alterations to the relative ratios of transcripts annotated to the same gene, and generate novel 3' ends because of differential APA. The growth of G. lemaneiformis was inhibited by Cu stress, while this inhibition was alleviated by ACC treatment. RNA-Seq analysis further revealed that 211 differential alternative splicing (DAS) events and 142 DAS events was obtained in CK vs Cu and Cu vs Cu + ACC, respectively, suggesting that AS of functional genes could be regulated by Cu stress and ACC. Compared with Cu stress, the expression of transcripts with DAS events mainly involved in the carbon fixation in photosynthetic organisms and oxidative phosphorylation pathway was upregulated in Cu + ACC treatment, revealing that ACC alleviated the growth inhibition by Cu stress by increasing carbon fixation and oxidative phosphorylation.ConclusionsOur results provide the first comprehensive picture of the full-length transcriptome and posttranscriptional mechanism in red macroalgae, including transcripts that appeared in the presence of common abiotic stresses and phytohormones, which will improve the gene annotations of Gracilariopsis and contribute to the study of gene regulation in this important cultivated seaweed.

Dataset Information

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

Publications

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

Similar Datasets

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