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

Leung, Szi Kay; Jeffries, Aaron R.; Castanho, Isabel; Jordan, B; Moore, Karen; Davies, Jonathan; Dempster, Emma; Bray, Nicholas John; O’Neill, Paul; Tseng, Elizabeth; Ahmed, Zeshan; Collier, David A.; Jeffery, Erin; Prabhakar, Shyam; Schalkwyk, Leonard; Jops, Connor; Gandal, Michael J.; Sheynkman, Gloria; Hannon, Eilís; Mill, Jonathan

doi:10.1016/j.celrep.2021.110022

Cited by 94 publications

(90 citation statements)

References 76 publications

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“…3i ). Similarly to recent bulk PacBio RNA sequencing of human cortex 34 , detected genes plateaued at ~12,000. For initiating reverse transcription, simulations suggest that poly(dT) priming captures entire polyadenylated molecules.…”

Section: Resultsmentioning

confidence: 79%

Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Hardwick

Joglekar

et al. 2022

Nat Biotechnol

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Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.

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Section: Resultsmentioning

confidence: 79%

Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Hardwick

Joglekar

et al. 2022

Nat Biotechnol

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“…To our knowledge, full-length transcriptomic data are not yet available for most organisms, especially not for mammals. One earlier study reported even lower number of genes and transcripts in human and mouse brain, but overall comparable quality [30]. Previously, mainly short-read RNA-sequencing was used for transcriptomic analysis.…”

Section: Deciphering Full-length High-fidelity Transcriptome With Dis...mentioning

confidence: 97%

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

et al. 2022

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Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction. Results Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice. Conclusions We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.

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“…These results suggest more efforts for annotating the chicken genome is needed in the future. The human genome is considered to be better-annotated than farm animals’, while 36.4% of full-length transcripts identified by long-read Iso-Seq methods are classified as “novel” in human cortex tissue (Leung et al, 2021). Using the same approach, another study also reported 17 to 55% of novel isoforms in human breast cancer samples (Veiga et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

A comprehensive prediction of transcript isoforms in 19 chicken tissues by Oxford Nanopore long-read sequencing

Guan

Halstead

Islas‐Trejo

et al. 2022

Preprint

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To comprehensively identify and annotate transcript isoforms in the chicken genome, we generated Nanopore long-read sequencing data from a diverse set of 19 chicken tissues comprising 68 samples collected from experimental line 6 × line 7 F1 adult males and females. More than 23.8 million reads with mean read length of 790 bases and average quality of 18.2 were generated. The annotation and subsequent filtering resulted in identification of 55,382 transcripts with mean length of 1,700 bases at 40,547 loci, representing ∼1.4 transcripts per locus. Among them, we predicted 30,967 potential coding transcripts at 19,461 loci and 16,495 potential lncRNA transcripts at 15,512 loci. Compared to reference annotations, we found 52% of annotated transcripts could partially to fully match while 47% were novel and potentially transcribed from lncRNA loci. Based on our annotation, we quantified transcript expression across tissues and found brain tissues (i.e. cerebellum, cortex) expressed highest number of transcripts and loci. The further tissue specificity revealed that ∼22% of the transcripts displaying tissue specificity. Of them, the reproductive tissues (i.e. testis, ovary) contained the most tissue-specific transcripts. Despite sequencing 68 transcriptomes derived from 19 tissues, still ∼20% of Ensembl reference loci were not detected. This suggests that including additional samples from different cell types, developmental and physiological conditions, is needed to fully annotate the chicken genome. The application of Nanopore sequencing transcriptomes in this study demonstrated the usefulness of long-read data in discovering additional novel loci (e.g., lncRNA loci) and resolving complex transcripts (e.g., the longest transcript for the TTN locus).

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

Cited by 94 publications

References 76 publications

Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

A comprehensive prediction of transcript isoforms in 19 chicken tissues by Oxford Nanopore long-read sequencing

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