Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: May 10, 2024

Abstract The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length sequencing to several kilobases, thereby facilitating identification alternative splicing events and isoform expressions. Recently, numerous computational tools for detection using long-read data have been developed. Nevertheless, there remains a deficiency in comparative studies that systemically evaluate performance these tools, which are implemented with different algorithms, under various simulations encompass potential influencing factors. In this study, we conducted benchmark analysis thirteen methods nine capable identifying structures from RNA-seq data. We evaluated their performances simulated data, represented diverse platforms generated by an in-house simulator, RNA sequins (sequencing spike-ins) as well experimental Our findings demonstrate IsoQuant highly effective tool LRS, Bambu StringTie2 also exhibiting strong performance. These results offer valuable guidance future research on ongoing improvement LRS

Language: Английский

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Clinical validation of RNA sequencing for Mendelian disorder diagnostics

The American Journal of Human Genetics, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Language: Английский

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Detection of mRNA Transcript Variants

Genes, Journal Year: 2025, Volume and Issue: 16(3), P. 343 - 343

Published: March 16, 2025

Most eukaryotic genes express more than one mature mRNA, defined as transcript variants. This complex phenomenon arises from various mechanisms, such using alternative transcription start sites and post-transcriptional processing events. The resulting variants can lead to synthesizing proteins that possess distinct functional domains or may even generate noncoding RNAs, each with unique roles in cellular processes. generation of these is not merely a random occurrence; it cell-type specific varies developmental stages, aging processes, pathogenesis diseases. highlights the biological significance regulating gene expression their potential impact on functionality. Despite importance, investigating has been hampered by challenges associated detecting expression. review article addresses advancements molecular techniques Traditional methods RT-PCR RT-qPCR easily detect known primers target exons Other like RACE-PCR hybridization-based methods, including Northern blotting, RNase protection assays, microarrays, have also utilized Nevertheless, RNA sequencing (RNA-Seq) emerged powerful technique for identifying variants, especially those previously unknown sequences. effectiveness variant detection depends approach precision data analysis. By understanding strengths weaknesses laboratory technique, researchers develop effective strategies mRNA ability will be crucial our comprehensive regulation implications diversity contexts.

Language: Английский

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Transcriptomics in the era of long-read sequencing

Nature Reviews Genetics, Journal Year: 2025, Volume and Issue: unknown

Published: March 28, 2025

Language: Английский

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Enhancing novel isoform discovery: leveraging nanopore long-read sequencing and machine learning approaches

Briefings in Functional Genomics, Journal Year: 2024, Volume and Issue: 23(6), P. 683 - 694

Published: Aug. 19, 2024

Long-read sequencing technologies can capture entire RNA transcripts in a single read, reducing the ambiguity constructing and quantifying transcript models comparison to more common earlier methods, such as short-read sequencing. Recent improvements accuracy of long-read have expanded scope for novel splice isoform detection also enabled far accurate reconstruction complex splicing patterns transcriptomes. Additionally, incorporation advancements machine learning deep algorithms bioinformatic software significantly improved reliability transcriptomic studies. However, there is lack consensus on what tools pipelines produce most precise consistent results. Thus, this review aims discuss compare performance available methods discovery with technologies, 25 being presented. Furthermore, intends demonstrate need developing standard analytical pipelines, tools, model conventions

Language: Английский

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Understanding isoform expression by pairing long-read sequencing with single-cell and spatial transcriptomics

Genome Research, Journal Year: 2024, Volume and Issue: 34(11), P. 1735 - 1746

Published: Nov. 1, 2024

RNA isoform diversity, produced via alternative splicing, and usage of transcription start poly(A) sites, results in varied transcripts being derived from the same gene. Distinct isoforms can play important biological roles, including by changing sequences or expression levels protein products. The first single-cell approaches to sequencing—and later, spatial approaches—which are now widely used for identification differentially expressed genes, rely on short reads offer ability transcriptomically compare different cell types but limited their measure differential expression. More recently, long-read sequencing methods have been combined with technologies order characterize In this review, we provide an overview emergence discuss challenges associated implementation these interpretation data. We opportunities they understanding relationships between distinct variable elements transcript molecules highlight some ways which isoforms’ roles development pathology. Single-nucleus sequencing, a special case approach, is also discussed. attempt cover both limitations significant potential expanding our still-limited isoforms.

Language: Английский

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Discovering the hidden function in fungal genomes

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Sept. 19, 2024

Language: Английский

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Long-read RNA sequencing can probe organelle genome pervasive transcription

Briefings in Functional Genomics, Journal Year: 2024, Volume and Issue: 23(6), P. 695 - 701

Published: June 17, 2024

Abstract 40 years ago, organelle genomes were assumed to be streamlined and, perhaps, unexciting remnants of their prokaryotic past. However, the field genomics has exposed an unparallel diversity in genome architecture (i.e. size, structure, and content). The transcription these eccentric can just as elaborate – are pervasively transcribed into a plethora RNA types. while protein-coding genes known produce polycistronic transcripts that undergo heavy posttranscriptional processing, nature noncoding transcriptomes is still poorly resolved. Here, we review how wet-lab experiments second-generation sequencing data short reads) have been useful determine certain types RNAs, particularly RNAs. We then explain third-generation (long-read) RNA-Seq represent new frontier transcriptomics. show public repositories (e.g. NCBI SRA) already contain enough for inter-phyla comparative studies argue biologists benefit from such data. discuss prospects using publicly available organelle-focused examine challenges approach. highlight lack comprehensive database dedicated genomics/transcriptomics major impediment development with implications basic applied science.

Language: Английский

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Clinical validation of RNA sequencing for Mendelian disorder diagnostics

medRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 17, 2024

Summary Despite rapid advancements in clinical sequencing, over half of diagnostic evaluations still lack definitive results. RNA-seq has shown promise research settings for bridging this gap by providing essential functional data accurate interpretation sequencing However, despite advanced pipelines, translation not yet been realized. We have developed and validated a test CLIA laboratory individuals with suspected genetic disorders who existing or concurrent comprehensive DNA testing. This processes patient RNA samples from fibroblasts blood derives interpretations based on the analytical detection outliers gene expressions splicing patterns. The validation involves 150 samples, including benchmark, negative, positive samples. provisional expression benchmarks using short-read long-read HG002 lymphoblastoid sample produced Genome Bottle Consortium. Our achieved sensitivity specificity higher than 99% against benchmarks. For validation, we first established reference ranges each junction distributions our control data. then evaluated performance outlier-based pipeline previously identified findings Undiagnosed Diseases Network project. 19 20 both fibroblast highlighted limitations test. study provides paradigm necessary resources independent laboratories to validate

Language: Английский

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Long-read RNA sequencing: a transformative technology for exploring transcriptome complexity in human diseases

Molecular Therapy, Journal Year: 2024, Volume and Issue: 33(3), P. 883 - 894

Published: Nov. 19, 2024

Long-read RNA sequencing (RNA-seq) is emerging as a powerful and versatile technology for studying human transcriptomes. By enabling the end-to-end of full-length transcripts, long-read RNA-seq opens up avenues investigating various species features that cannot be reliably interrogated by standard short-read methods. In this review, we present an overview RNA-seq, delineating its strengths over well summarizing recent advances in experimental computational approaches to boost power long-read-based transcriptomics. We describe wide range applications highlight expanding role foundational exploring transcriptome variations diseases.

Language: Английский

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