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Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes.

ABSTRACT: Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

SUBMITTER: Zhu C

PROVIDER: S-EPMC8270901 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms

Project description:Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that accurately quantifies transcript isoforms in their entire length from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. By establishing an isoform-differential expression test, our approach revealed that 11 of these genes displayed no detectable change in overall RNA expression. However, significant differences in the expression of specific isoforms in these genes was observed. These isoform specific effects demonstrate the need of analyzing RNA isoform expression levels rather than total gene expression levels.

2021-06-16 | E-MTAB-7334 | biostudies-arrayexpress

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Project description:Background:Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs. Methods:We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors. Results:A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5' and 3' untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants. Discussion:The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

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Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method.

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Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

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Single molecule, near full-length genome sequencing of dengue virus.

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Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris.

Project description:Background:Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. Findings:We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. Conclusions:We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

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Cellular, transcriptomic and isoform heterogeneity of breast cancer cell line revealed by full-length single-cell RNA sequencing.

Project description:Tumor heterogeneity is generated through a combination of genetic and epigenetic mechanisms, the latter of which plays an important role in the generation of stem like cells responsible for tumor formation and metastasis. Although the development of single cell transcriptomic technologies holds promise to deconvolute this complexity, a number of these techniques have limitations including drop-out and uneven coverage, which challenge the further delineation of tumor heterogeneity. We adopted deep and full-length single-cell RNA sequencing on Fluidigm's Polaris platform to reveal the cellular, transcriptomic, and isoform heterogeneity of SUM149, a triple negative breast cancer (TNBC) cell line. We first validate the quality of the TNBC sequencing data with the sequencing data from erythroleukemia K562 cell line as control. We next scrutinized well-defined marker genes for cancer stem-like cell to identify different cell populations. We then profile the isoform expression data to investigate the heterogeneity of alternative splicing patterns. Though classified as triple-negative breast cancer, the SUM149 stem cells show heterogeneous expression of marker receptors (ER, PR, and HER2) across the cells. We identified three cell populations that express patterns of stemness: epithelial-mesenchymal transition (EMT) cancer stem cells (CSCs), mesenchymal-epithelial transition (MET) CSCs and Dual-EMT-MET CSCs. These cells also manifested a high level of heterogeneity in alternative splicing patterns. For example, CSCs have shown different expression patterns of the CD44v6 exon, as well as different levels of truncated EGFR transcripts, which may suggest different potentials for proliferation and invasion among cancer stem cells. Our study identified features of the landscape of previously underestimated cellular, transcriptomic, and isoform heterogeneity of cancer stem cells in triple-negative breast cancers.

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

Project description: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.

| S-EPMC8609283 | biostudies-literature

Benchmarking full-length transcript single cell mRNA sequencing protocols

Project description:Here we present a performance evaluation of three different plate-based scRNA-seq protocols. Two commercially available kits; NEBNext Single Cell/ Low Input RNA Library Prep Kit (NEB), SMART-seq HT kit (Takara), and non-commercially available protocol Genome & transcriptome sequencing (G&T). We evaluated each kit based upon sensitivity, reproducibility, ease of use and price point per cell. This evaluation is specifically relevant for implementation of scRNA-seq in e.g. diagnostics, requiring high sensitivity in regards of gene detection, high reproducibility between samples, minimum hands on time and smooth sample preparation for technicians, as well as keeping a reasonable price point per patient. G&T protocol delivered the highest detection of genes per single cell, at the absolute lowest price point. Takara's kit delivered likewise high gene detection per single cell, and high reproducibility between sample, however at the absolute highest price points. Here we present pros and cons of each protocol, sequencing breast cancer cell line T47D.

2020-08-01 | GSE155506 | GEO

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