Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing

Hargreaves, Adam D; Mulley, John F.

doi:10.7717/peerj.1441

Cited by 36 publications

(26 citation statements)

References 45 publications

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“…This is particularly appealing when rapid identification is required for a biological sample (Borsting & Morling, ; Greninger et al., ; Hoenen et al., ; Kalianski et al., ; Quick et al., , ). However, what is much less desirable is the low per base accuracy of MinION™ (~90%: Hargreaves & Mulley, ; Ip, Loose, Tyson, & de Cesare, ; Mikheyev & Tin, ; Sović et al., ) which generates many bioinformatics challenges. These challenges are increasingly overcome by new bioinformatic pipelines.…”

Section: Introductionmentioning

confidence: 99%

A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

Srivathsan

Baloğlu

Wang³

et al. 2018

Molecular Ecology Resources

105

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DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory and is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION™ reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION™ barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION™ barcodes have an accuracy of 99.3%-100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be

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Section: Introductionmentioning

confidence: 99%

A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

Srivathsan

Baloğlu

Wang³

et al. 2018

Molecular Ecology Resources

105

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“…The Oxford Nanopore Technologies (ONT) MinION sequencer is a portable device that is based on single molecule sequencing technology that provides reads of unprecedented length by performing voltage driven molecule translocations through small nanosensors 26 . Although the MinION platform has been most useful for interrogating viral and bacterial genomes, recently it has been applied for analyzing cDNA in both targeted as well as genome-wide approaches 17,[27][28][29][30] . Taking advantage of its unprecedented read length, we wanted to interrogate single-cell transcriptomes of mouse B1a cells by sequencing full-length cDNA molecules using the ONT MinION sequencer.…”

Section: Introductionmentioning

confidence: 99%

Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells

Byrne

Beaudin

Cole

et al. 2017

Preprint

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Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single cell level. Short-read RNAseq, while the current standard for gene expression quantification, is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigated whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencing technology (ONT RNAseq) would be able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After successfully benchmarking our experimental and computational approaches on a mixture of synthetic transcripts, we analyzed individual murine B1a cells using a new cellular indexing strategy. Using the Mandalorion analysis pipeline we developed, we identified thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identified hundreds of genes expressed across B1a cells that displayed multiple complex isoforms, including several B cell specific surface receptors and the antibody heavy chain (IGH) locus. Our results show that not only can we identify complex isoforms, but also quantify their expression, at the single cell level.

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“…Аналогично SMRT-секвенированию, нанопоровое секвенирование используется в гибридном секвенировании как технология, "комплементарная" SGS [89][90][91][92]. Применительно к анализу транскриптома, корректировка длинных "чтений" с помощью коротких (с использованием программы proovread) позволила снизить ошибку нанопорового секвенирования с 12% до 0-2% [93]. Нанопоровое секвенирование также было применено для секвенирования транскриптома de novo [94], для чего авторами был разработан алгоритм анализа длинных "чтений", основанный на их кластеризации на основе схожести без обращения к референсному геному или транскриптому, получивший название CARNAC-LR (Clustering coefficient-based Acquisition of RNA Communities in Long Reads).…”

Section: анализ транскриптома с использованием нанопорового секвенироunclassified

Prospects for the use of third generation sequencers for quantitative profiling of transcriptome

Radko

Курбатов

Ptitsyn

et al. 2018

BMCRM

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Transcriptome profiling is widely employed to analyze transcriptome dynamics when studying various biological processes at the cell and tissue levels. Unlike the second generation sequencers, which sequence relatively short fragments of nucleic acids, the third generation DNA/RNA sequencers developed by biotechnology companies “PacBio” and “Oxford Nanopore Technologies” allow one to sequence transcripts as single molecules and may be considered as potential molecular counters capable to measure the number of copies of each transcript with high throughput, sensitivity, and specificity. In the present review, the features of single molecule sequencing technologies offered by “PacBio” and “Oxford Nanopore Technologies” are considered alongside with their utility for transcriptome analysis, including the analysis of transcript isoforms. The prospects and limitations of the single molecule sequencing technology in application to quantitative transcriptome profiling are also discussed.

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Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing

Cited by 36 publications

References 45 publications

A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells

Prospects for the use of third generation sequencers for quantitative profiling of transcriptome

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