Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

Nature Methods, Год журнала: 2024, Номер 21(7), С. 1349 - 1363

Опубликована: Июнь 7, 2024

Abstract The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, consortium generated over 427 million sequences from complementary DNA direct RNA datasets, encompassing human, mouse manatee species. Developers utilized these data address challenges in transcript isoform detection, quantification de novo detection. study revealed that libraries with longer, more accurate produce transcripts than those increased read depth, whereas greater depth improved accuracy. In well-annotated genomes, tools based on reference demonstrated best performance. Incorporating additional orthogonal replicate samples is advised when aiming detect rare novel or using reference-free approaches. This collaborative offers a benchmark current practices provides direction future method development

Язык: Английский

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Importance of pre-mRNA splicing and its study tools in plants

Advanced Biotechnology, Год журнала: 2024, Номер 2(1)

Опубликована: Фев. 8, 2024

Alternative splicing (AS) significantly enriches the diversity of transcriptomes and proteomes, playing a pivotal role in physiology development eukaryotic organisms. With continuous advancement high-throughput sequencing technologies, an increasing number novel transcript isoforms, along with factors related to their associated functions, are being unveiled. In this review, we succinctly summarize compare different mechanisms across prokaryotes eukaryotes. Furthermore, provide extensive overview recent progress various studies on AS covering developmental stages diverse plant species response abiotic stresses. Additionally, discuss modern techniques for studying functions quantification transcripts, as well protein products. By integrating genetic studies, quantitative methods, omics techniques, can discover isoforms functional factors, thereby enhancing our understanding roles modes species.

Язык: Английский

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Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июль 27, 2023

Abstract The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. consortium generated over 427 million sequences from cDNA and direct RNA datasets, encompassing human, mouse, manatee species, using different protocols sequencing platforms. These data were utilized by developers address challenges in transcript isoform detection quantification, as well de novo identification. study revealed that libraries with longer, more accurate produce transcripts than those increased read depth, whereas greater depth improved quantification accuracy. In well-annotated genomes, tools based on reference demonstrated best performance. When aiming detect rare novel or when reference-free approaches, incorporating additional orthogonal replicate samples are advised. This collaborative offers a benchmark current practices provides direction future method development

Язык: Английский

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Single-cell and spatial transcriptomics: Bridging current technologies with long-read sequencing

Molecular Aspects of Medicine, Год журнала: 2024, Номер 96, С. 101255 - 101255

Опубликована: Фев. 17, 2024

Язык: Английский

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Advances in long-read single-cell transcriptomics

Human Genetics, Год журнала: 2024, Номер 143(9-10), С. 1005 - 1020

Опубликована: Май 24, 2024

Abstract Long-read single-cell transcriptomics (scRNA-Seq) is revolutionizing the way we profile heterogeneity in disease. Traditional short-read scRNA-Seq methods are limited their ability to provide complete transcript coverage, resolve isoforms, and identify novel transcripts. The protocols developed for long-read sequencing platforms overcome these limitations by enabling characterization of full-length techniques initially suffered from comparatively poor accuracy compared short read scRNA-Seq. However, with improvements accuracy, accessibility, cost efficiency, long-reads gaining popularity field This review details advances scRNA-Seq, an emphasis on library preparation downstream bioinformatics analysis tools.

Язык: Английский

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Discovery of Novel Protein-Coding and Long Non-coding Transcripts in Distinct Regions of the Human Brain

Journal of Molecular Neuroscience, Год журнала: 2025, Номер 75(1)

Опубликована: Март 6, 2025

Recent improvements in the accuracy of long-read sequencing (LRS) technologies have expanded scope for novel transcriptional isoform discovery. Additionally, these advancements improved precision transcript quantification, enabling a more accurate reconstruction complex splicing patterns and transcriptomes. Thus, this project aims to take advantage analytical developments discovery analysis RNA isoforms human brain. A set was compiled using three bioinformatic tools, quantifying their expression across eight replicates cerebellar hemisphere, five frontal cortex, six putamen. By taking subset consistent all methods, 170 highly confident curated downstream analysis. This consisted 104 messenger RNAs (mRNAs) 66 long non-coding (lncRNAs) isoforms. The detailed structure, expression, potential encoded proteins mRNA BambuTx321 been further described as an exemplary representative. tissue-specific [mean counts per million (CPM) 5.979] lncRNA, BambuTx1299, hemisphere observed. Overall, has identified annotated several diverse tissues brain, providing insights into investigating functional roles. contributed comprehensive understanding brain's transcriptomic landscape applications basic research.

Язык: Английский

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Transcriptomics in the era of long-read sequencing

Nature Reviews Genetics, Год журнала: 2025, Номер unknown

Опубликована: Март 28, 2025

Язык: Английский

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Detecting haplotype-specific transcript variation in long reads with FLAIR2

Genome biology, Год журнала: 2024, Номер 25(1)

Опубликована: Июль 2, 2024

Abstract Background RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these variants a variety of diseases. Aberrant splicing and single nucleotide (SNVs) have been demonstrated to alter transcript stability, localization, function. In particular, the upregulation ADAR, an enzyme that mediates adenosine-to-inosine editing, previously linked increase invasiveness lung adenocarcinoma cells associated with regulation. Despite functional importance studying SNVs, use short-read limited community’s ability interrogate both forms variation simultaneously. Results We employ long-read sequencing technology obtain full-length sequences, elucidating cis-effects on changes at molecule level. develop computational workflow augments FLAIR, tool calls isoform models expressed data, integrate variant isoforms bear them. generate nanopore data high sequence accuracy from H1975 without knockdown ADAR . apply our identify key inosine associations help clarify prominence tumorigenesis. Conclusions Ultimately, we find approach provides valuable insight toward characterizing relationship between patterns.

Язык: Английский

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Evaluation of strategies for evidence-driven genome annotation using long-read RNA-seq

Genome Research, Год журнала: 2024, Номер unknown, С. gr.279864.124 - gr.279864.124

Опубликована: Дек. 23, 2024

While the production of a draft genome has become more accessible due to long-read sequencing, annotation these new genomes not been developed at same pace. Long-read RNA sequencing (lrRNA-seq) offers promising solution for enhancing gene annotation. In this study, we explore how platforms, Oxford Nanopore R9.4.1 chemistry or PacBio Sequel II CCS, and data processing methods influence evidence-driven using long reads. Incorporating transcripts into our pipeline significantly outperformed traditional methods, such as ab initio predictions short-read-based annotations. We applied strategy nonmodel species, Florida manatee, compared results existing At loci level, both annotations were highly concordant, with 90% agreement. However, transcript agreement was only 35%. identified 4,906 novel loci, represented by 5,707 isoforms, 64% isoforms matching known sequences in other mammalian species. Overall, findings underscore importance high-quality curated models combination effective

Язык: Английский

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Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host

PLoS Pathogens, Год журнала: 2023, Номер 19(6), С. e1011204 - e1011204

Опубликована: Июнь 8, 2023

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little known about the viral genes that mediate transmission, mostly due to a lack natural virus-host model systems. Marek's disease devastating herpesviral chickens caused by virus (MDV) and an excellent study skin-tropic transmission. Like varicella zoster causes chicken pox humans, only site where infectious cell-free MD virions are efficiently produced epithelial skin cells, requirement host-to-host Here, we enriched heavily infected feather follicle cells live measure both transcription protein expression using combined short- long-read RNA sequencing LC/MS-MS bottom-up proteomics. Enrichment previously unseen breadth depth peptide sequencing. We confirmed translation 84 at high confidence (1% FDR) correlated relative abundance with levels. Using proteogenomic approach, most well-characterized spliced transcripts identified novel, abundant isoform 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning reads, high-quality junction-spanning identification. peptides representing alternative start codon usage several putative novel microORFs 5' ends two core genes, pUL47 ICP4, along strong evidence independent capsid scaffold pUL26.5. animal system examine gene provides robust, efficient, meaningful way validating results gathered from cell culture

Язык: Английский

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