Dataset Information

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

ABSTRACT: Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a reference Klebsiella pneumoniae isolate demonstrated ?99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum ?-lactamase (ESBL)-producing Escherichia coli and K. pneumoniae isolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.

SUBMITTER: Lemon JK

PROVIDER: S-EPMC5703817 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

Lemon Jamie K JK Khil Pavel P PP Frank Karen M KM Dekker John P JP

Journal of clinical microbiology 20171011 12

Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid s ...[more]

PMID: 29021151

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Project description:ObjectiveWe aimed to evaluate the performance of nanopore amplicon sequencing detection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples.MethodWe carried out a single-center, prospective cohort study in a Wuhan hospital and collected a total of 86 clinical samples, including 54 pharyngeal swabs, 31 sputum samples, and 1 fecal sample, from 86 patients with coronavirus disease 2019 (COVID-19) from Feb 20 to May 15, 2020. We performed parallel detection with nanopore-based genome amplification and sequencing (NAS) on the Oxford Nanopore Technologies (ONT) minION platform and routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). In addition, 27 negative control samples were detected using the two methods. The sensitivity and specificity of NAS were evaluated and compared with those of RT-qPCR.ResultsThe viral read number and reference genome coverage were both significantly different between the two groups of samples, and the latter was a better indicator for SARS-CoV-2 detection. Based on the reference genome coverage, NAS revealed both high sensitivity (96.5%) and specificity (100%) compared with RT-qPCR (80.2 and 96.3%, respectively), although the samples had been stored for half a year before the detection. The total time cost was less than 15 h, which was acceptable compared with that of RT-qPCR (∼2.5 h). In addition, the reference genome coverage of the viral reads was in line with the cycle threshold value of RT-qPCR, indicating that this number could also be used as an indicator of the viral load in a sample. The viral load in sputum might be related to the severity of the infection, particularly in patients within 4 weeks after onset of clinical manifestations, which could be used to evaluate the infection.ConclusionOur results showed the high sensitivity and specificity of the NAS method for SARS-CoV-2 detection compared with RT-qPCR. The sequencing results were also used as an indicator of the viral load to display the viral dynamics during infection. This study proved the wide application prospect of nanopore sequencing detection for SARS-CoV-2 and may more knowledge about the clinical characteristics of COVID-19.

Dataset Information

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

Publications

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

Similar Datasets

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