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Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

ABSTRACT: Alternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.

SUBMITTER: Leung SK

PROVIDER: S-EPMC8609283 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

Leung Szi Kay SK Jeffries Aaron R AR Castanho Isabel I Jordan Ben T BT Moore Karen K Davies Jonathan P JP Dempster Emma L EL Bray Nicholas J NJ O'Neill Paul P Tseng Elizabeth E Ahmed Zeshan Z Collier David A DA Jeffery Erin D ED Prabhakar Shyam S Schalkwyk Leonard L Jops Connor C Gandal Michael J MJ Sheynkman Gloria M GM Hannon Eilis E Mill Jonathan J

Cell reports 20211101 7

Alternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporatin ...[more]

PMID: 34788620

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Project description:The full-length double-strand cDNA sequencing, one of the RNA-Seq methods, is a powerful method used to investigate the transcriptome status of a gene of interest, such as its transcription level and alternative splicing variants. Furthermore, full-length double-strand cDNA sequencing has the advantage that it can create a library from a small amount of sample and the library can be applied to long-read sequencers in addition to short-read sequencers. Nevertheless, one of our previous studies indicated that the full-length double-strand cDNA sequencing yields non-specific genomic DNA amplification, affecting transcriptome analysis, such as transcript quantification and alternative splicing analysis. In this study, it was confirmed that it is possible to produce the RNA-Seq library from only genomic DNA and that the full-length double-strand cDNA sequencing of genomic DNA yielded non-specific genomic DNA amplification. To avoid non-specific genomic DNA amplification, two methods were examined, which are the DNase I-treated full-length double-strand cDNA sequencing and poly(A) capture full-length double-strand cDNA sequencing. Contrary to expectations, the non-specific genomic DNA amplification was increased and the number of the detected expressing genes was reduced in DNase I-treated full-length double-strand cDNA sequencing. On the other hand, in the poly(A) capture full-length double-strand cDNA sequencing, the non-specific genomic DNA amplification was significantly reduced, accordingly the accuracy and the number of detected expressing genes and splicing events were increased. The expression pattern and percentage spliced in index of splicing events were highly correlated. Our results indicate that the poly(A) capture full-length double-strand cDNA sequencing improves transcript quantification accuracy and the detection ability of alternative splicing events. It is also expected to contribute to the determination of the significance of DNA variants to splicing events.

Project description:IntroductionPeanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut.MethodsIn this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform.Results and discussionAs a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the "response to stress", "chloroplast part", and "transcription factor activity" were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops.

Dataset Information

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

Publications

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

Similar Datasets

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