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

Isabelle Heifetz Ament,

Nicole DeBruyne,

Feng Wang

et al.

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

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation DOI
Mikhail Kolmogorov, Kimberley Billingsley, Mira Mastoras

et al.

Nature Methods, Journal Year: 2023, Volume and Issue: 20(10), P. 1483 - 1492

Published: Sept. 14, 2023

Language: Английский

Citations

81
Applications of long-read sequencing to Mendelian genetics DOI Creative Commons
Francesco Mastrorosa, Danny E. Miller, Evan E. Eichler

et al.

Genome Medicine, Journal Year: 2023, Volume and Issue: 15(1)

Published: June 14, 2023

Advances in clinical genetic testing, including the introduction of exome sequencing, have uncovered molecular etiology for many rare and previously unsolved disorders, yet more than half individuals with a suspected disorder remain after complete evaluation. A precise diagnosis may guide treatment plans, allow families to make informed care decisions, permit participate N-of-1 trials; thus, there is high interest developing new tools techniques increase solve rate. Long-read sequencing (LRS) promising technology both increasing rate decreasing amount time required diagnosis. Here, we summarize current LRS technologies, give examples how they been used evaluate complex variation identify missing variants, discuss future applications LRS. As costs continue decrease, will find additional utility space fundamentally changing pathological variants are discovered eventually acting as single-data source that can be interrogated multiple times service.

Language: Английский

Citations

58
Nanopore sequencing of 1000 Genomes Project samples to build a comprehensive catalog of human genetic variation DOI Creative Commons
Jonas A. Gustafson, Sophia B Gibson, Nikhita Damaraju

et al.

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

Published: March 7, 2024

Less than half of individuals with a suspected Mendelian condition receive precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest using long-read sequencing (LRS) to streamline genomic testing, but the absence control datasets for variant filtering prioritization has made tertiary analysis LRS challenging. To address this, 1000 Genomes Project ONT Sequencing Consortium aims generate from at least 800 samples. Our goal is use identify broader spectrum variation so we may improve our understanding normal patterns human variation. Here, present first 100 samples, representing all 5 superpopulations 19 subpopulations. These sequenced an average depth coverage 37x sequence read N50 54 kbp, high concordance previous studies identifying single nucleotide indel variants outside homopolymer regions. Using multiple structural (SV) callers, 24,543 high-confidence SVs per genome, including shared private likely disrupt gene function as well pathogenic expansions within disease-associated repeats that were not detected short reads. Evaluation methylation signatures revealed expected known imprinted loci, samples skewed X-inactivation patterns, novel differentially methylated All raw data, processed summary statistics are publicly available, providing valuable resource genetics community discover SVs.

Language: Английский

Citations

26
HiPhase: jointly phasing small, structural, and tandem repeat variants from HiFi sequencing DOI Creative Commons
James Holt, Christopher T. Saunders, William J. Rowell

et al.

Bioinformatics, Journal Year: 2024, Volume and Issue: 40(2)

Published: Jan. 25, 2024

Abstract Motivation In diploid organisms, phasing is the problem of assigning alleles at heterozygous variants to one two haplotypes. Reads from PacBio HiFi sequencing provide long, accurate observations that can be used as basis for both calling and variants. reads also excel larger classes variation, such structural or tandem repeat However, current tools typically only phase small variants, leaving unphased. Results We developed HiPhase, a tool jointly phases SNVs, indels, structural, The main benefits HiPhase are (i) dual mode allele assignment detecting large (ii) novel application A*-algorithm phasing, (iii) logic allowing blocks span breaks caused by alignment issues around reference gaps homozygous deletions. our assessment, produced an average block NG50 480 kb with 929 switchflip errors fully phased 93.8% genes, improving over state art. Additionally, includes innate multi-threading, statistics gathering, concurrent output generation. Availability implementation available source code pre-compiled Linux binary user guide https://github.com/PacificBiosciences/HiPhase.

Language: Английский

Citations

13
Severus: accurate detection and characterization of somatic structural variation in tumor genomes using long reads DOI Creative Commons

Ayse Keskus,

Asher Bryant, Tanveer Ahmad

et al.

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

Published: March 26, 2024

Abstract Most current studies rely on short-read sequencing to detect somatic structural variation (SV) in cancer genomes. Long-read offers the advantage of better mappability and long-range phasing, which results substantial improvements germline SV detection. However, long-read detection methods do not generalize well analysis SVs tumor genomes with complex rearrangements, heterogeneity, aneuploidy. Here, we present Severus: a method for accurate different types using phased breakpoint graph approach. To benchmark various short- methods, sequenced five tumor/normal cell line pairs Illumina, Nanopore, PacBio platforms; this Severus showed highest F1 scores (harmonic mean precision recall) as compared methods. We then applied three clinical cases pediatric cancer, demonstrating concordance known genetic findings revealing clinically relevant cryptic rearrangements missed by standard genomic panels.

Language: Английский

Citations

9
A Hitchhiker's Guide to long-read genomic analysis DOI
Medhat Mahmoud, Daniel Paiva Agustinho, Fritz J. Sedlazeck

et al.

Genome Research, Journal Year: 2025, Volume and Issue: 35(4), P. 545 - 558

Published: April 1, 2025

Over the past decade, long-read sequencing has evolved into a pivotal technology for uncovering hidden and complex regions of genome. Significant cost efficiency, scalability, accuracy advancements have driven this evolution. Concurrently, novel analytical methods emerged to harness full potential long reads. These enabled milestones such as first fully completed human genome, enhanced identification understanding genomic variants, deeper insights interplay between epigenetics variation. This mini-review provides comprehensive overview latest developments in DNA analysis, encompassing reference-based de novo assembly approaches. We explore entire workflow, from initial data processing variant calling annotation, focusing on how these improve our ability interpret wide array variants. Additionally, we discuss current challenges, limitations, future directions field, offering detailed examination state-of-the-art bioinformatics sequencing.

Language: Английский

Citations

1
Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation DOI Creative Commons
Mikhail Kolmogorov, Kimberley Billingsley, Mira Mastoras

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 15, 2023

Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due a combination being too expensive, scalable enough, or error-prone. Here, we develop an efficient and wet lab computational protocol for Oxford Nanopore Technologies (ONT) long-read that seeks provide genuine alternative large-scale genomics projects. We applied our cell lines brain tissue samples part pilot project NIH Center Alzheimer's Related Dementias (CARD). Using single PromethION flow cell, can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains be difficult inside homopolymers tandem repeats, is comparable calls elsewhere. Further, discover structural variants state-of the-art methods involving Pacific Biosciences HiFi trio information (but lower cost greater throughput). ONT based phasing, then combine phase small megabase scales. Our also produces highly accurate, haplotype-specific methylation calls. Overall, this makes projects feasible; currently used sequence thousands brain-based genomes CARD initiative. software open-source integrated pipelines generating phased variant assemblies.

Language: Английский

Citations

19
MethPhaser: methylation-based long-read haplotype phasing of human genomes DOI Creative Commons
Yilei Fu, Sergey Aganezov, Medhat Mahmoud

et al.

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

Published: June 22, 2024

The assignment of variants across haplotypes, phasing, is crucial for predicting the consequences, interaction, and inheritance mutations a key step in improving our understanding phenotype disease. However, phasing limited by read length stretches homozygosity along genome. To overcome this limitation, we designed MethPhaser, method that utilizes methylation signals from Oxford Nanopore Technologies to extend Single Nucleotide Variation (SNV)-based phasing. We demonstrate haplotype-specific methylations extensively exist Human genomes advent long-read technologies enabled direct report signals. For ONT R9 R10 cell line data, increase phase N50 78%-151% at accuracy 83.4-98.7% assess impact tissue purity random due inactivation, also applied MethPhaser on blood samples 4 patients, still showing improvements over SNV-only further improves HLA multiple other medically relevant genes, how interact phenotypes. concept can be extended non-human diploid genomes. available https://github.com/treangenlab/methphaser .

Language: Английский

Citations

8
3-hour genome sequencing and targeted analysis to rapidly assess genetic risk DOI Creative Commons

Miranda Pg Zalusky,

Jonas A. Gustafson, Stephanie C. Bohaczuk

et al.

Genetics in Medicine Open, Journal Year: 2024, Volume and Issue: 2, P. 101833 - 101833

Published: Jan. 1, 2024

Rapid genetic testing in the critical care setting may guide diagnostic evaluation, direct therapies, and help families providers make informed decisions about goals of care. We tested whether a simplified DNA extraction library preparation process would enable us to perform ultra-rapid assessment risk for Mendelian condition, based on information from an affected sibling, using long-read genome sequencing targeted analysis.

Language: Английский

Citations

6
Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes DOI Creative Commons
Kieran O’Neill, Erin Pleasance,

Jeremy Fan

et al.

Cell Genomics, Journal Year: 2024, Volume and Issue: 4(11), P. 100674 - 100674

Published: Oct. 14, 2024

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This from POG program Marathon Hope Cancer Centres Network includes DNA RNA short-read sequence data, analytics, clinical information. We show potential long-read sequencing for resolving complex cancer-related structural variants, viral integrations, extrachromosomal circular DNA. Long-range phasing facilitates discovery allelically differentially methylated regions (aDMRs) allele-specific expression, including recurrent aDMRs in cancer genes RET CDKN2A. Germline promoter methylation MLH1 can be directly observed Lynch syndrome. Promoter BRCA1 RAD51C is likely driver behind homologous recombination deficiency where no coding mutation was found. demonstrates applications precision medicine available as resource developing analytical approaches this technology.

Language: Английский

Citations

6