Next-Generation Sequencing and Emerging Technologies

Seminars in Thrombosis and Hemostasis, Год журнала: 2019, Номер 45(07), С. 661 - 673

Опубликована: Май 16, 2019

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation (NGS) emerging methodologies. NGS has tremendously improved output while being more time cost-efficient in comparison to Sanger sequencing. The describe short-read approaches, such as by synthesis, ion semiconductor sequencing, nanoball Third-generation long-read now promises overcome many limitations ability reliably resolve repeat sequences large genomic rearrangements. By combining complementary methods massively parallel DNA greater insight into biological context disease mechanisms is possible. Emerging methodologies, advances nanopore technology, situ nucleic acid microscopy-based will continue evolution area. These new hold potential applications hematological disorders, promise precision personalized medical care future.

Язык: Английский

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Decoding the epitranscriptional landscape from native RNA sequences

Nucleic Acids Research, Год журнала: 2020, Номер 49(2), С. e7 - e7

Опубликована: Июль 13, 2020

Abstract Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct of native using Oxford Nanopore Technologies (ONT) can allow for directly detecting the base modifications, although these modifications might appear as errors. The percent Error Specific Bases (%ESB) was higher than unmodified RNA, which enabled detection ribonucleotide sites. Based %ESB differences, we developed bioinformatic tool, epitranscriptional landscape inferring from glitches ONT signals (ELIGOS), that based various types synthetic modified applied rRNA mRNA. ELIGOS able accurately predict known classes methylation sites (AUC > 0.93) in rRNAs Escherichiacoli, yeast, human cells, either vitro transcription background error model, mimics systematic direct reference. well-known DRACH/RRACH motif localized identified, consistent previous studies, differential analysis study impact m6A methyltransferase comparing wild type knockouts yeast mouse cells. Lastly, DRACH could also be identified mRNA three cell lines. at level individual resolution. In summary, have software package uncover modifications.

Язык: Английский

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Direct RNA sequencing enables m⁶A detection in endogenous transcript isoforms at base-specific resolution

RNA, Год журнала: 2019, Номер 26(1), С. 19 - 28

Опубликована: Окт. 17, 2019

Direct RNA sequencing holds great promise for the de novo identification of modifications at single-coordinate resolution; however, interpretation raw output to discover modified bases remains a challenge. Using Oxford Nanopore's direct technology, we developed random forest classifier trained using experimentally detected N 6 -methyladenosine (m A) sites within DRACH motifs. Our software MINES A Identification Nanopore Sequencing) assigned m methylation status more than 13,000 previously unannotated in endogenous HEK293T transcripts and identified 40,000 with isoform-level resolution human mammary epithelial cell line. These displayed sensitivity writer, METTL3, eraser, ALKBH5, respectively. (https://github.com/YeoLab/MINES.git) enables annotation single coordinate–level from nanopore sequencing.

Язык: Английский

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Neutrophil extracellular traps mediate m⁶A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells

International Journal of Biological Sciences, Год журнала: 2022, Номер 18(8), С. 3337 - 3357

Опубликована: Янв. 1, 2022

Neutrophil extracellular traps (NETs) production is a major strategy employed by polymorphonuclear neutrophils (PMNs) to fight against microbes. NETs have been implicated in the pathogenesis of various lung injuries, although few studies explored sepsis-associated acute injury (SI-ALI). Here, we demonstrate contribution pathology ALI inducing ferroptosis alveolar epithelial cells. Using both vitro and vivo studies, our findings show enhanced accumulation patients mice, as well closely related upregulation ferroptosis, induction which depends on METTL3-induced m6A modification GPX4. CLP-induced mouse model established with METTL3-/- versus WT addition METTL3 knockout overexpression vitro, elucidated confirmed critical role NETs-induced ALI. These support for subsequent

Язык: Английский

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RNA methylation and cancer treatment

Pharmacological Research, Год журнала: 2021, Номер 174, С. 105937 - 105937

Опубликована: Окт. 13, 2021

To this date, over 100 different types of RNA modification have been identified. Methylation species has emerged as a critical regulator transcript expression. methylation and its related downstream signaling pathways are involved in plethora biological processes, including cell differentiation, sex determination stress response, others. It is catalyzed by the methyltransferases, demethylated demethylases (FTO ALKBH5) read binding protein (YTHDF1 IGF2BP1). Increasing evidence indicates that process closely connected to cancer proliferation, cellular stress, metastasis, immune response. And becoming promising targets therapy. This review outlines relationship between cancer, some FTO inhibitors treatment.

Язык: Английский

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Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing

Nucleic Acids Research, Год журнала: 2019, Номер 47(20), С. e126 - e126

Опубликована: Авг. 16, 2019

Abstract Methylation of guanosine on position N7 (m7G) internal RNA positions has been found in all domains life and have implicated human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection modifications at nucleotide resolution. In our method, modified are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription sequenced. We detect increased mutation rates the reduced control samples taking possibility sequencing/alignment error into account use replicates calculate statistical significance based log likelihood ratio tests. show that m7G-MaP-seq efficiently detects known rRNA mutational up 25% map a previously uncharacterised evolutionarily conserved modification 1581 Arabidopsis thaliana SSU rRNA. Furthermore, identify budding yeast, arabidopsis tRNAs demonstrate occurs before tRNA splicing. do not find any evidence for being other small RNA, such miRNA, snoRNA sRNA, including Let-7e. Likewise, depth analysis mRNA from E. coli or yeast cells did modifications.

Язык: Английский

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Detection of m6A from direct RNA sequencing using a multiple instance learning framework

Nature Methods, Год журнала: 2022, Номер 19(12), С. 1590 - 1598

Опубликована: Ноя. 10, 2022

Abstract RNA modifications such as m6A methylation form an additional layer of complexity in the transcriptome. Nanopore direct sequencing can capture this information raw current signal for each molecule, enabling detection using supervised machine learning. However, experimental approaches provide only site-level training data, whereas modification status single molecule is missing. Here we present m6Anet, a neural-network-based method that leverages multiple instance learning framework to specifically handle missing read-level labels data. m6Anet outperforms existing computational methods, shows similar accuracy approaches, and generalizes with high different cell lines species without retraining model parameters. In addition, demonstrate captures underlying stoichiometry, which be used approximate differences rates. Overall, offers tool transcriptome-wide identification quantification from run sequencing.

Язык: Английский

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Naturally occurring modified ribonucleosides

Wiley Interdisciplinary Reviews - RNA, Год журнала: 2020, Номер 11(5)

Опубликована: Апрель 16, 2020

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as "fifth" ribonucleotide in 1951. Since then, ever-increasing number and complexity modified have been found viruses throughout all three domains life. Such modifications can be simple methylations, hydroxylations, or thiolations, complex ring closures, glycosylations, acylations, aminoacylations, unusual incorporation selenium. While initially transfer ribosomal RNAs, also exist messenger RNAs noncoding RNAs. Modifications profound cellular outcomes at various levels, such altering structure being essential for cell survival organism viability. aberrant presence absence lead to human disease, ranging from cancer metabolic developmental illnesses Hoyeraal-Hreidarsson syndrome, Bowen-Conradi Williams-Beuren syndrome. In this review article, we summarize characterization 143 currently known by describing their taxonomic distributions, enzymes that generate modifications, any implications processes, structure, disease. We highlight areas active research, specific contain a particular type modification well methodologies used identify novel modifications. This article is categorized under: Processing > Editing Modification.

Язык: Английский

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Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA

Nature Methods, Год журнала: 2019, Номер 16(12), С. 1281 - 1288

Опубликована: Сен. 23, 2019

Язык: Английский

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METTL1 promotes hepatocarcinogenesis via m⁷G tRNA modification‐dependent translation control

Clinical and Translational Medicine, Год журнала: 2021, Номер 11(12)

Опубликована: Дек. 1, 2021

Abstract Background N 7 ‐methylguanosine (m G) modification is one of the most common transfer RNA (tRNA) modifications in humans. The precise function and molecular mechanism m G tRNA hepatocellular carcinoma (HCC) remain poorly understood. Methods prognostic value expression level methyltransferase complex components methyltransferase‐like protein‐1 (METTL1) WD repeat domain 4 (WDR4) HCC were evaluated using clinical samples TCGA data. biological functions mechanisms progression studied vitro vivo cell culture, xenograft model, knockin knockout mouse models. reduction cleavage sequencing (TRAC‐seq), polysome profiling polyribosome‐associated mRNA methods used to study levels modification, translation efficiency. Results METTL1 WDR4 are elevated associated with advanced tumour stages poor patient survival. Functionally, silencing or inhibits proliferation, migration invasion, while forced wild‐type but not its catalytic dead mutant promotes progression. Knockdown reduces decreases G‐modified cells. Mechanistically, METTL1‐mediated target mRNAs higher frequencies G‐related codons. Furthermore, studies Mettl1 conditional mice reveal essential physiological hepatocarcinogenesis hydrodynamics transfection model. Conclusions Our work reveals new insights into role misregulated liver cancer provides basis for diagnosis treatment.

Язык: Английский

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