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Rapid and sensitive single-sample viral metagenomics using Nanopore Flongle sequencing


Pichler, Ian; Schmutz, Stefan; Ziltener, Gabriela; Zaheri, Maryam; Kufner, Verena; Trkola, Alexandra; Huber, Michael (2023). Rapid and sensitive single-sample viral metagenomics using Nanopore Flongle sequencing. Journal of Virological Methods, 320:114784.

Abstract

The ability of viral metagenomic Next-Generation Sequencing (mNGS) to unbiasedly detect nucleic acids in a clinical sample is a powerful tool for advanced diagnosis of viral infections. When clinical symptoms do not provide a clear differential diagnosis, extensive laboratory testing with virus-specific PCR and serology can be replaced by a single viral mNGS analysis. However, widespread diagnostic use of viral mNGS is thus far limited by long sample-to-result times, as most protocols rely on Illumina sequencing, which provides high and accurate sequencing output but is time-consuming and expensive. Here, we describe the development of an mNGS protocol based on the more cost-effective Nanopore Flongle sequencing with decreased turnaround time and lower, yet sufficient sequencing output to provide sensitive virus detection. Sample preparation (6 h) and sequencing (2 h) times are substantially reduced compared to Illumina mNGS and allow detection of DNA/RNA viruses at low input (up to 33-38 cycle threshold of specific qPCR). Although Flongles yield lower sequencing output, direct comparison with Illumina mNGS on diverse clinical samples showed similar results. Collectively, the novel Nanopore mNGS approach is specifically tailored for use in clinical diagnostics and provides a rapid and cost-effective mNGS strategy for individual testing of severe cases.

Abstract

The ability of viral metagenomic Next-Generation Sequencing (mNGS) to unbiasedly detect nucleic acids in a clinical sample is a powerful tool for advanced diagnosis of viral infections. When clinical symptoms do not provide a clear differential diagnosis, extensive laboratory testing with virus-specific PCR and serology can be replaced by a single viral mNGS analysis. However, widespread diagnostic use of viral mNGS is thus far limited by long sample-to-result times, as most protocols rely on Illumina sequencing, which provides high and accurate sequencing output but is time-consuming and expensive. Here, we describe the development of an mNGS protocol based on the more cost-effective Nanopore Flongle sequencing with decreased turnaround time and lower, yet sufficient sequencing output to provide sensitive virus detection. Sample preparation (6 h) and sequencing (2 h) times are substantially reduced compared to Illumina mNGS and allow detection of DNA/RNA viruses at low input (up to 33-38 cycle threshold of specific qPCR). Although Flongles yield lower sequencing output, direct comparison with Illumina mNGS on diverse clinical samples showed similar results. Collectively, the novel Nanopore mNGS approach is specifically tailored for use in clinical diagnostics and provides a rapid and cost-effective mNGS strategy for individual testing of severe cases.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Virology
Uncontrolled Keywords:Clinical diagnostics; Flongle flow cells; Nanopore sequencing; Viral metagenomics
Language:English
Date:October 2023
Deposited On:15 Jan 2024 14:08
Last Modified:30 Jun 2024 01:37
Publisher:Elsevier
ISSN:0166-0934
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.jviromet.2023.114784
PubMed ID:37516367
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)