MinoTour, real-time monitoring and analysis for Nanopore Sequencers

Munro, Rory; Santos, Roberto; Payne, Alexander; Forey, Teri; Osei, Solomon; Holmes, Nadine; Loose, Matthew

doi:10.1101/2021.09.10.459783

Cited by 3 publications

(3 citation statements)

References 14 publications

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“…One of the unique strengths of ONT-based sequencing methods is that, beyond the standard approach of analyzing sequencing runs once they are completed, many library metrics can be derived in real-time. This is starting to get exploited in clinical and metagenomics assays with tools like SURPIrt 34 or with more powerful tools like MinoTour 35 . The PLNK tool we developed here controls basecalling by the guppy5 basecaller, C3POa processing, mappy-based alignment and on-target estimation for enrichment analysis.…”

Section: Discussionmentioning

confidence: 99%

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

Zee

Deng

Adams

et al. 2021

Preprint

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High-throughput short-read sequencing has taken on a central role in research and diagnostics. Literally hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the inertia associated with the research enterprise as a whole have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the long-read ONT MinION by circularizing and rolling circle amplifying the short library molecules using the R2C2 method. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into millions of high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

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Section: Discussionmentioning

confidence: 99%

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

Zee

Deng

Adams

et al. 2021

Preprint

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show abstract

“…Then the first primary mapping for any read was determined and mappings binned into windows along the genome such that on average each bin contains 100 reads. Runs were monitored in real-time using minoTour (https://github.com/LooseLab/minotourapp/; commit: 1f9c678), providing coverage statistics, mappings and estimates of copy number variation in real-time 16 . During real-time analysis reads were mapped to Chm13 telomere-to-telomere assembly 20,21 .…”

Section: Library Preparation Sequencing and Analysismentioning

confidence: 99%

“…Adaptive sampling increases read count as a consequence of rejecting molecules once they are confidently mapped to an off-target region. We therefore developed a simple approach to bin read counts across the genome such that, on average, each bin would contain 100 reads, and monitored this in real-time using our minoTour tool 16 . For each barcoded sample changes in copy number are immediately apparent and can be visualised using any change point detection approach, here we use Ruptures (Figure 5) 17 .…”

Section: Main Textmentioning

confidence: 99%

Barcode aware adaptive sampling for GridION and PromethION Oxford Nanopore sequencers

Payne¹,

Munro²,

Holmes

et al. 2021

Preprint

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Adaptive sampling enables selection of individual molecules from sequencing libraries, a unique property of nanopore sequencing. Here we develop our adaptive sampling tool readfish to become "barcode-aware" enabling selection of different targets within barcoded samples or filtering out individual barcodes. We show that multiple human genomes can be assessed for copy number and structural variation on a single sequencing flow cell using sample specific customised target panels.

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Real-time monitoring and analysis of SARS-CoV-2 nanopore sequencing with minoTour

Munro

Holmes

Moore

et al. 2021

Preprint

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Motivation: The ongoing SARS-CoV-2 pandemic has demonstrated the utility of real-time analysis of sequencing data, with a wide range of databases and resources for analysis now available. Here we show how the real-time nature of Oxford Nanopore Technologies sequencers can accelerate consensus generation, lineage and variant status assignment. We exploit the fact that multiplexed viral sequencing libraries quickly generate sufficient data for the majority of samples, with diminishing returns on remaining samples as the sequencing run progresses. We demonstrate methods to determine when a sequencing run has passed this point in order to reduce the time required and cost of sequencing. Results: We extended MinoTour, our real-time analysis and monitoring platform for nanopore sequencers, to provide SARS-CoV2 analysis using ARTIC network pipelines. We additionally developed an algorithm to predict which samples will achieve sufficient coverage, automatically running the ARTIC medaka informatics pipeline once specific coverage thresholds have been reached on these samples. After testing on run data, we find significant run time savings are possible, enabling flow cells to be used more efficiently and enabling higher throughput data analysis. The resultant consensus genomes are assigned both PANGO lineage and variant status as defined by Public Health England. Samples from within individual runs are used to generate phylogenetic trees incorporating optional background samples as well as summaries of individual SNPs. As minoTour uses ARTIC pipelines, new primer schemes and pathogens can be added to allow minoTour to aid in real-time analysis of pathogens in the future.

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MinoTour, real-time monitoring and analysis for Nanopore Sequencers

Cited by 3 publications

References 14 publications

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

Barcode aware adaptive sampling for GridION and PromethION Oxford Nanopore sequencers

Real-time monitoring and analysis of SARS-CoV-2 nanopore sequencing with minoTour

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