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Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Published on: December 22, 2014
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Improvements in metagenomic virus detection by simple pretreatment methods.
Anna S Fomsgaard1,2, Morten Rasmussen1, Katja Spiess1
1Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen, Denmark.
Journal of Clinical Virology Plus
|March 22, 2023
Summary
This study presents a new, unbiased method for detecting RNA and DNA viruses at the point of care using Nanopore sequencing. The rapid assay allows for same-day results, improving pathogen detection and reducing the need for specialized labs.
Area of Science:
- Virology
- Genomics
- Bioinformatics
Background:
- Early pathogen detection is crucial for public health and preventing outbreaks.
- Current methods often require specialized laboratories and sample transport, delaying results.
- Identifying unknown pathogens necessitates unbiased detection strategies.
Purpose of the Study:
- To develop an unbiased, field-deployable assay for detecting RNA and DNA viruses.
- To optimize pretreatment methods for enhancing viral read proportions in clinical samples.
- To demonstrate the assay's efficacy for identifying SARS-CoV-2, HPV, and MCV.
Main Methods:
- Metagenomic Nanopore sequencing of clinical samples.
- Optimization of nucleic acid pretreatment, including DNase I treatment and filtration.
- Testing with human clinical samples containing SARS-CoV-2, HPV, and MCV.
- RT-qPCR for determining viral loads (CT-values) of SARS-CoV-2.
Main Results:
- DNase I pretreatment and filtration increased SARS-CoV-2 reads >500-fold.
- High-confidence virus detection and variant identification were achieved.
- The assay successfully detected DNA viruses (HPV, MCV) and RNA viruses (SARS-CoV-2) in clinical samples.
Conclusions:
- A rapid, field-adaptable Nanopore sequencing assay for unbiased virus detection was developed.
- The optimized protocol enables same-day results for RNA and DNA viruses.
- This method has the potential to revolutionize point-of-care diagnostics by eliminating sample transport and cold storage needs.
