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Rapid semirandom virus diagnostics using Nanopore amplicon sequencing

English title Rapid semirandom virus diagnostics using Nanopore amplicon sequencing
Applicant Huber Michael
Number 190648
Funding scheme Spark
Research institution Institut für Medizinische Virologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Infectious Diseases
Start/End 01.02.2020 - 31.07.2022
Approved amount 77'600.00
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Keywords (6)

Metagenomic sequencing; Amplicon sequencing; Random amplification; Syndromic panels; Nanopore sequencing; Virus diagnostics

Lay Summary (German)

Lead
Die modernen molekularbiologischen Verfahren in der Virusdiagnostik sind sensitiv, schnell und kostengünstig. Für jeden Erreger muss jedoch ein eigener Test bereitgestellt werden. Wir erarbeiten in diesem Projekt eine neue und schnelle metagenomische Sequenzierung zum direkten Nachweis eines breiten Spektrums von Viren in klinischen Proben.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Die metagenomische Sequenzierung ermöglicht den offenen, unvoreingenommenen Nachweis von theoretisch jedem Virus oder anderen Krankheitserreger. Die Methode ist aber nach wie vor zeitaufwendig und weniger empfindlich als die spezifische PCR. Das Ziel dieses Projekts ist es, diese Nachteile zu beheben. Unser Ansatz basiert auf einer Kombination aus zufälliger und virusspezifischer Amplifikation des Virusgenoms. Wir werden vier syndromische Amplifikationspanel etablieren, die Viren repräsentieren, die mit klinisch relevanten Krankheitsmustern assoziiert sind. Die Sequenzierung erfolgt mit einer neuartigen und schnellen amplikonbasierten Methode auf der Nanopore Plattform.

 

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Eine frühzeitige und vollständige Diagnose von Infektionskrankheiten ist wichtig, um eine rechtzeitige Behandlung zu ermöglichen und das Risiko von Ansteckungen zu reduzieren. Die metagenomische Sequenzierung kann bei Infektionen mit vielen in Frage kommenden Erregern (z.B. Meningitis/Enzephalitis, Atemwegsinfektionen, fieberhafte Erkrankungen) die Routinediagnostik unterstützen und zur raschen Diagnostik beitragen.

Direct link to Lay Summary Last update: 11.12.2019

Responsible applicant and co-applicants

Employees

Name Institute

Associated projects

Number Title Start Funding scheme
200550 Dynamic reference indexes for selective sequencing with application to diagnostics 01.01.2022 Project funding (Div. I-III)

Abstract

Tests commonly applied in routine virus diagnostics are tailored to be highly sensitive, rapid and cost effective. The disadvantage of these methods is, however, that each pathogen requires its own specific assay. Routine diagnostic laboratories can only afford to offer assays for the most frequent pathogens. Therefore, the etiology of infection in many cases remains unknown and often multiple tests against different potential pathogens need to be performed to reach a conclusive diagnosis.Recent development of high-throughput sequencing has provided unprecedented opportunities in direct pathogen detection. Today, metagenomic sequencing allows for open, unbiased genomic detection of virtually any viral or other pathogen in clinical samples. However, the workflows are still time consuming and less sensitive than specific PCR.Here, we propose to establish and validate an original strategy to overcome the limitations of turnaround time and sensitivity. The approach is based on semirandom anchored PCR amplification using a combination of random and virus-specific primers. We will generate four syndromic primer libraries representing virus panels associated with clinically relevant disease patterns. By means of a novel and fast library preparation, amplicon-based Nanopore sequencing will be applied for rapid diagnostics of a broad range of viral targets in clinical samples in a single assay.We will probe this new approach first with control samples spiked with viruses and will then validated it on a large repository of clinical samples with known virus infections. We expect a higher sensitivity due to the inclusion of specific primers for amplification. The key differentiator to other approaches will be the direct amplicon sequencing library preparation and the real-time analysis using Nanopore sequencing technology.Rapid, early and complete diagnosis of infections is highly desirable as it will allow timely treatment of the infected patients and reduce risks of nosocomial spread. Metagenomic sequencing can avoid multiple testing for broad differential diagnoses (e.g. meningitis/encephalitis, respiratory infections, febrile illnesses) and prevent unnecessary antibiotic treatment.We think our project is a small, but promising plan that probes an unconventional and bold semirandom PCR amplification approach. It further combines this approach with state-of-the-art Nanopore sequencing technology and is therefore ideally suited for funding by the Spark program.
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