Advances in nanopore direct RNA sequencing

Nature Methods, Journal Year: 2022, Volume and Issue: 19(10), P. 1160 - 1164

Published: Oct. 1, 2022

Nanopore direct RNA sequencing (DRS) reads continuous native strands. Early adopters have used this technology to document nucleotide modifications and 3′ polyadenosine tails on strands without added chemistry steps. Individual ranging in length from 70 26,000 nucleotides been sequenced. In our opinion, broader acceptance of nanopore DRS by molecular biologists cell will be accelerated higher basecall accuracy lower input requirements.

Language: Английский

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Benchmarking of Nanopore R10.4 and R9.4.1 flow cells in single-cell whole-genome amplification and whole-genome shotgun sequencing

Computational and Structural Biotechnology Journal, Journal Year: 2023, Volume and Issue: 21, P. 2352 - 2364

Published: Jan. 1, 2023

Third-generation sequencing can be used in human cancer genomics and epigenomic research. Oxford Nanopore Technologies (ONT) recently released R10.4 flow cell, which claimed an improved read accuracy compared to R9.4.1 cell. To evaluate the benefits defects of cell for profiling on MinION devices, we non-small-cell lung-carcinoma line HCC78 construct libraries both single-cell whole-genome amplification (scWGA) shotgun sequencing. The reads were benchmarked terms accuracy, variant detection, modification calling, genome recovery rate with next generation (NGS) reads. results highlighted that outperforms reads, achieving a higher modal over 99.1%, superior variation lower false-discovery (FDR) methylation comparable rate. achieve high yields scWGA ONT platform as NGS, recommended multiple displacement modified T7 endonuclease Ⅰ cutting procedure promising method. In addition, provided possible solution filter likely false positive sites among whole region by using result negative control. Our study is first benchmark cells clarifying capacity genomic within single A method together calling benefit researchers who work third-generation

Language: Английский

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Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

PLoS Computational Biology, Journal Year: 2023, Volume and Issue: 19(3), P. e1010905 - e1010905

Published: March 2, 2023

A perfect bacterial genome assembly is one where the assembled sequence an exact match for organism’s genome—each replicon complete and contains no errors. While this has been difficult to achieve in past, improvements long-read sequencing, assemblers, polishers have brought assemblies within reach. Here, we describe our recommended approach assembling a perfection using combination of Oxford Nanopore Technologies long reads Illumina short reads: Trycycler assembly, Medaka polishing, Polypolish short-read followed by other polishing tools manual curation. We also discuss potential pitfalls might encounter when challenging genomes, provide online tutorial with sample data ( github.com/rrwick/perfect-bacterial-genome-tutorial ).

Language: Английский

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Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction

Microbial Genomics, Journal Year: 2023, Volume and Issue: 9(1)

Published: Jan. 10, 2023

Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) short-read (Illumina) datasets. Being able to use nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released new flowcell (R10.4) chemistry (Kit12), which reportedly generate per-read accuracies rivalling those Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa Staphylococcus aureus, ONT's R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy assembly each modality, considering the impact different basecalling models, used assemblers, sequencing depth, duplex versus simplex reads. 'Super accuracy' (sup) basecalled R10.4 reads - in particular high could robustly reconstruct without However, per-run yield generated our hands standard protocols was low <10 %), substantial implications cost throughput if relying on enable genome reconstruction. In addition, recovery small plasmids best-performing long-read assembler (Flye) inconsistent. combined sup holds promise as singular technology genomes, but (Illumina+R9.4.1 hac) remains highest throughput, most robust, cost-effective approach fully these genomes.

Language: Английский

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Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 41(7), P. 1018 - 1025

Published: Jan. 2, 2023

Nanopore sequencers can select which DNA molecules to sequence, rejecting a molecule after analysis of small initial part. Currently, selection is based on predetermined regions interest that remain constant throughout an experiment. Sequencing efforts, thus, cannot be re-focused likely contributing most experimental success. Here we present BOSS-RUNS, algorithmic framework and software generate dynamically updated decision strategies. We quantify uncertainty at each genome position with real-time updates from data already observed. For fragment, decide whether the expected decrease in it would provide warrants fully sequencing it, thus optimizing information gain. BOSS-RUNS mitigates coverage bias between within members microbial community, leading improved variant calling; for example, low-coverage sites species 1% abundance were reduced by 87.5%, 12.5% more single-nucleotide polymorphisms detected. Such data-driven are applicable many scenarios, such as enriching increased divergence or low coverage, reducing time-to-answer.

Language: Английский

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The newest Oxford Nanopore R10.4.1 full-length 16S rRNA sequencing enables the accurate resolution of species-level microbial community profiling

Applied and Environmental Microbiology, Journal Year: 2023, Volume and Issue: 89(10)

Published: Oct. 6, 2023

ABSTRACT The long-read amplicon provides a species-level solution for the community. With improvement of nanopore flowcells, accuracy Oxford Nanopore Technologies (ONT) R10.4.1 has been substantially enhanced, with an average approximately 99%. To evaluate its effectiveness on amplicons, three types microbiomes were analyzed by 16S ribosomal RNA (hereinafter referred to as “16S”) sequencing using Novaseq, Pacbio sequel II, and PromethION platforms (R9.4.1 R10.4.1) in current study. We showed error rate, recall, precision, bias index mock sample. rate ONT was greatly reduced, better recall case synthetic Meanwhile, different environmental samples, analysis resulted composition similar data. found that classification tools databases influence Based these results, we conclude can also be used application samples. IMPORTANCE supplies community solution. Due high early on, it not frequently studies. introduced flowcell Q20+ reagent achieve more than 99% technology advanced. However, there no published study performance commercial sequencers flowcells or impact taxonomy In this study, investigated (rRNA) R10.4.1). sample, displayed index. observed is significantly lower, especially when deletions are involved. First foremost, Pacific Bioscience reveal microbiome This shows full-length rRNA sequences allow species identification microbiota.

Language: Английский

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Applications of long-read sequencing to Mendelian genetics

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

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A high-quality genome compendium of the human gut microbiome of Inner Mongolians

Nature Microbiology, Journal Year: 2023, Volume and Issue: 8(1), P. 150 - 161

Published: Jan. 5, 2023

Language: Английский

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SemiBin2: self-supervised contrastive learning leads to better MAGs for short- and long-read sequencing

Bioinformatics, Journal Year: 2023, Volume and Issue: 39(Supplement_1), P. i21 - i29

Published: June 1, 2023

Metagenomic binning methods to reconstruct metagenome-assembled genomes (MAGs) from environmental samples have been widely used in large-scale metagenomic studies. The recently proposed semi-supervised method, SemiBin, achieved state-of-the-art results several environments. However, this required annotating contigs, a computationally costly and potentially biased process.We propose SemiBin2, which uses self-supervised learning learn feature embeddings the contigs. In simulated real datasets, we show that achieves better than SemiBin1 SemiBin2 outperforms other binners. Compared SemiBin1, can 8.3-21.5% more high-quality bins requires only 25% of running time 11% peak memory usage short-read sequencing samples. To extend long-read data, also ensemble-based DBSCAN clustering algorithm, resulting 13.1-26.3% second best binner for data.SemiBin2 is available as open source software at https://github.com/BigDataBiology/SemiBin/ analysis scripts study be found https://github.com/BigDataBiology/SemiBin2_benchmark.

Language: Английский

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Nanopore DNA sequencing technologies and their applications towards single-molecule proteomics

Nature Chemistry, Journal Year: 2024, Volume and Issue: 16(3), P. 314 - 334

Published: March 1, 2024

Language: Английский

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