Nanopore Technology and Its Applications in Gene Sequencing

Lin, Bo; Hui, Jianan; Mao, Hongju

doi:10.3390/bios11070214

Cited by 126 publications

(86 citation statements)

References 117 publications

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“…MinION's main disadvantage is a relatively low read accuracy compared with the highly accurate sequencing of Sanger and Illumina methods, the estimated error rate being between 5 and 15% [30][31][32] despite its constant improvement. Therefore, the analysis of ONT sequence reads is a complex task, particularly when variant detection is required.…”

Section: Discussionmentioning

confidence: 99%

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Rosa

Brandtner²,

Mancini

et al. 2021

Water

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The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.

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Section: Discussionmentioning

confidence: 99%

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Rosa

Brandtner²,

Mancini

et al. 2021

Water

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“…The portability of the MinION device, coupled with its fast turnaround time, turns it into a promising tool to be used in clinical practice for early diagnosis, treatment, therapeutic monitoring, etc. [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Oxford Nanopore MinION Direct RNA-Seq for Systems Biology

Pyatnitskiy

Arzumanian

Radko

et al. 2021

Biology

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Long-read direct RNA sequencing developed by Oxford Nanopore Technologies (ONT) is quickly gaining popularity for transcriptome studies, while fast turnaround time and low cost make it an attractive instrument for clinical applications. There is a growing interest to utilize transcriptome data to unravel activated biological processes responsible for disease progression and response to therapies. This trend is of particular interest for precision medicine which aims at single-patient analysis. Here we evaluated whether gene abundances measured by MinION direct RNA sequencing are suited to produce robust estimates of pathway activation for single sample scoring methods. We performed multiple RNA-seq analyses for a single sample that originated from the HepG2 cell line, namely five ONT replicates, and three replicates using Illumina NovaSeq. Two pathway scoring methods were employed—ssGSEA and singscore. We estimated the ONT performance in terms of detected protein-coding genes and average pairwise correlation between pathway activation scores using an exhaustive computational scheme for all combinations of replicates. In brief, we found that at least two ONT replicates are required to obtain reproducible pathway scores for both algorithms. We hope that our findings may be of interest to researchers planning their ONT direct RNA-seq experiments.

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“…It is conceivable that the ONT and PacBio platforms are unsuitable for measuring interstrand crosslinks because they are both sequencing single-stranded DNA. Therefore, efforts and developments are on the way to use wider pores to perform sequencing of double-stranded DNA [109][110][111]. Crosslinks of DNA to whole proteins will probably not work with this new technology.…”

Section: Single Molecule Dna Sequencingmentioning

confidence: 99%

Current and Future Methodology for Quantitation and Site-Specific Mapping the Location of DNA Adducts

Boysen

Nookaew

2022

Toxics

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Formation of DNA adducts is a key event for a genotoxic mode of action, and their presence is often used as a surrogate for mutation and increased cancer risk. Interest in DNA adducts are twofold: first, to demonstrate exposure, and second, to link DNA adduct location to subsequent mutations or altered gene regulation. Methods have been established to quantitate DNA adducts with high chemical specificity and to visualize the location of DNA adducts, and elegant bio-analytical methods have been devised utilizing enzymes, various chemistries, and molecular biology methods. Traditionally, these highly specific methods cannot be combined, and the results are incomparable. Initially developed for single-molecule DNA sequencing, nanopore-type technologies are expected to enable simultaneous quantitation and location of DNA adducts across the genome. Herein, we briefly summarize the current methodologies for state-of-the-art quantitation of DNA adduct levels and mapping of DNA adducts and describe novel single-molecule DNA sequencing technologies to achieve both measures. Emerging technologies are expected to soon provide a comprehensive picture of the exposome and identify gene regions susceptible to DNA adduct formation.

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Nanopore Technology and Its Applications in Gene Sequencing

Cited by 126 publications

References 117 publications

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Oxford Nanopore MinION Direct RNA-Seq for Systems Biology

Current and Future Methodology for Quantitation and Site-Specific Mapping the Location of DNA Adducts

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