Nanopore sequencing technology and its applications

Zheng, Peijie; Zhou, Chuntao; Ding, Yuemin; Liu, Bin; Lu, Liuyi; Zhu, Feng; Duan, Shiwei

doi:10.1002/mco2.316

Cited by 9 publications

(3 citation statements)

References 334 publications

(771 reference statements)

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“…ONT Amplicon-Seq has several limitations that should be considered. First, the err rate of the ONT reads is still quite high [48]. Polymerase errors during amplification a another source of sequencing errors [32].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nanopore Amplicon Sequencing Allows Rapid Identification of Glutenin Allelic Variants in a Wheat Collection

Polkhovskaya,

Gruzdev,

Moskalev

et al. 2023

Agronomy

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Genetic variation in high molecular weight glutenin (HMW-GS) genes is tightly linked with the breadmaking quality of wheat. Hundreds of different alleles have been identified in HMW-GS genes worldwide. Such huge variability makes it difficult to distinguish them using conventional genotyping methods (for example, SDS-PAGE, SNP detection, etc.). Here, we exploited the nanopore amplicon sequencing technique (Amplicon-Seq) to uncover genetic variants distributed along the full-length sequence of six HMW-GSs, including the promoter and protein-coding regions. We analyzed 23 wheat accessions for allelic variants of HMW-GSs using the Amplicon-Seq and SDS-PAGE methods. We obtained sufficient (>50×) target gene coverage by ONT reads in just one hour. Using the obtained data, we identified numerous single nucleotide polymorphisms and InDels in the protein coding and promoter regions. Moreover, Amplicon-Seq allowed for the identification of new alleles (Glu-A1x1-T) of the Glu-1Ax gene that could not be recognized by SDS-PAGE. Collectively, our results showed that Amplicon-Seq is a rapid, multiplexed, and efficient method for high-throughput genotyping of full-length genes in large and complex genomes. This opens new avenues for the assessment of target gene variation to select novel alleles and create unique combinations of desirable traits in plant breeding programs.

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Section: Discussionmentioning

confidence: 99%

“…Secondly, primer design an PCR optimization are the most time-consuming and critical steps in ONT Amplicon-Se ONT Amplicon-Seq has several limitations that should be considered. First, the error rate of the ONT reads is still quite high [48]. Polymerase errors during amplification are another source of sequencing errors [32].…”

Section: Discussionmentioning

confidence: 99%

Nanopore Amplicon Sequencing Allows Rapid Identification of Glutenin Allelic Variants in a Wheat Collection

Polkhovskaya,

Gruzdev,

Moskalev

et al. 2023

Agronomy

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show abstract

“…One noteworthy alternative is long-read sequencing, such as Nanopore sequencing (Nanopore-seq). This technology, developed by Oxford Nanopore Technologies (ONT), has emerged as an innovative method for sequencing native long-read nucleic acids, including genomic DNA, cDNA, and RNA (Lu, Giordano, and Ning 2016, Wang et al 2021, Zheng et al 2023). The library preparation procedure involves straightforward steps, integrating a specific adapter at the end of the nucleic acid.…”

Section: Introductionmentioning

confidence: 99%

Real-time transcriptomic profiling in distinct experimental conditions

Butto,

Pastore,

Müller

et al. 2024

Preprint

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Nanopore-technology offers real-time sequencing opportunities, providing rapid access to sequenced data and allowing researchers to manage the sequencing process efficiently, resulting in cost-effective strategies. Here, we present focused case studies demonstrating the versatility of real-time transcriptomics analysis in rapid quality control for long-read RNA-seq. We illustrate its utility through three experimental setups: 1) transcriptome profiling of distinct human cellular populations, 2) identification of experimentally enriched transcripts, and 3) identification of experimentally manipulated genes (knockout and overexpression) in several yeast strains. We show how to perform multiple layers of quality control as soon as sequencing has started, addressing both the quality of the experimental and sequencing traits. Real-time quality control measures comprise assessing sample/condition variability and determining the number of identified genes per sample/condition. Furthermore, real-time differential gene/transcript expression analysis can be conducted at various time points post-sequencing initiation (PSI), revealing dynamic changes in gene/transcript expression between two conditions. Using real-time analysis, which occurs in parallel to the sequencing run, we identified differentially expressed genes/transcripts as early as 1-hour PSI. These changes were consistently observed throughout the entire sequencing process. We discuss the new possibilities offered by real-time data analysis, which have the potential to serve as a valuable tool for rapid and cost-effective quality checks in specific experimental settings and can be potentially integrated into clinical applications in the future.

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Pilot validation of on‐field STR typing and human identity testing by MinION nanopore sequencing

Luo,

Zhang,

et al. 2024

Electrophoresis

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Nanopore sequencing technology has broad application prospects in forensic medicine due to its small size, portability, fast speed, real‐time result analysis capabilities, single‐molecule sequencing abilities, and simple operation. Here, we demonstrate for the first time that nanopore sequencing platforms can be used to identify individuals in the field. Through scientific and reasonable design, a nanopore MinION MK1B device and other auxiliary devices are integrated into a portable detection box conducive to individual identification at the accident site. Individual identification of 12 samples could be completed within approximately 24 h by jointly detecting 23 short tandem repeat (STR) loci. Through double‐blinded experiments, the genotypes of 49 samples were successfully determined, and the accuracy of the STR genotyping was verified by the gold standard. Specifically, the typing success rate for 1150 genotypes was 95.3%, and the accuracy rate was 86.87%. Although this study focused primarily on demonstrating the feasibility of full‐process testing, it can be optimistically predicted that further improvements in bioinformatics workflows and nanopore sequencing technology will help enhance the feasibility of Oxford Nanopore Technologies equipment for real‐time individual identification at accident sites.

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Nanopore sequencing technology and its applications

Cited by 9 publications

References 334 publications

Nanopore Amplicon Sequencing Allows Rapid Identification of Glutenin Allelic Variants in a Wheat Collection

Nanopore Amplicon Sequencing Allows Rapid Identification of Glutenin Allelic Variants in a Wheat Collection

Real-time transcriptomic profiling in distinct experimental conditions

Pilot validation of on‐field STR typing and human identity testing by MinION nanopore sequencing

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