A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing.

Neuenschwander, Stefan Moritz; Terrazos Miani, Miguel Angel; Amlang, Heiko; Perroulaz, Carmen; Bittel, Pascal; Casanova, Carlo; Droz, Sara; Flandrois, Jean-Pierre; Leib, Stephen L.; Suter-Riniker, Franziska; Ramette, Alban (2020). A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing. Journal of clinical microbiology, 58(6) American Society for Microbiology 10.1128/JCM.00060-20

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Amplicon sequencing of 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories, and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations with the most significant being the inability to resolve mixed amplicons when closely related species are co-amplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read NGS technologies could solve the mixed amplicon issue, but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling flexible number of samples per run and adjustable sequencing time. Here we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (Long Read Consensus ANalysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN's behaviour when dealing with samples with known cross-contamination level. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability and availability of amplicon sequencing in diagnostic settings.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Mycobacteriology
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Research
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Infection Serology
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Clinical Microbiology
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > General Bacteriology

UniBE Contributor:

Neuenschwander, Stefan; Terrazos Miani, Miguel Angel; Amlang, Heiko; Perroulaz, Carmen; Bittel, Pascal; Casanova, Carlo; Droz, Sara Christine; Leib, Stephen; Suter, Franziska Marta and Ramette, Alban

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0095-1137

Publisher:

American Society for Microbiology

Language:

English

Submitter:

Stephen Leib

Date Deposited:

15 Jun 2020 15:28

Last Modified:

15 Jun 2020 15:37

Publisher DOI:

10.1128/JCM.00060-20

PubMed ID:

32229603

BORIS DOI:

10.7892/boris.144281

URI:

https://boris.unibe.ch/id/eprint/144281

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