Dataset Information

Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk.

ABSTRACT: Metagenomic sequencing with the Oxford Nanopore MinION sequencer offers potential for point-of-care testing of infectious diseases in clinical settings. To improve cost-effectiveness, multiplexing of several, barcoded samples upon a single flow cell will be required during sequencing. We generated a unique sequencing dataset to assess the extent and source of cross barcode contamination caused by multiplex MinION sequencing. Sequencing libraries for three different viruses, including influenza A, dengue, and chikungunya, were prepared separately and sequenced on individual flow cells. We also pooled the respective libraries and performed multiplex sequencing. We identified 0.056% of total reads in the multiplex sequencing data that were assigned to incorrect barcodes. Chimeric reads were the predominant source of this error. Our findings highlight the need for careful filtering of multiplex sequencing data before downstream analysis, and the trade-off between sensitivity and specificity that applies to the barcode demultiplexing methods.

SUBMITTER: Xu Y

PROVIDER: S-EPMC6156371 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk.

Xu Yifei Y Lewandowski Kuiama K Lumley Sheila S Pullan Steven S Vipond Richard R Carroll Miles M Foster Dona D Matthews Philippa C PC Peto Timothy T Crook Derrick D

Frontiers in microbiology 20180919

Metagenomic sequencing with the Oxford Nanopore MinION sequencer offers potential for point-of-care testing of infectious diseases in clinical settings. To improve cost-effectiveness, multiplexing of several, barcoded samples upon a single flow cell will be required during sequencing. We generated a unique sequencing dataset to assess the extent and source of cross barcode contamination caused by multiplex MinION sequencing. Sequencing libraries for three different viruses, including influenza A ...[more]

PMID: 30283430

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Project description:Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×-19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

Dataset Information

Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk.

Publications

Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk.

Similar Datasets

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