Improvements in metagenomic virus detection by simple pretreatment methods
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Improvements in metagenomic virus detection by simple pretreatment methods. / Fomsgaard, Anna S.; Rasmussen, Morten; Spiess, Katja; Fomsgaard, Anders; Belsham, Graham J.; Fonager, Jannik.
In: Journal of Clinical Virology Plus, Vol. 2, No. 4, 100120, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Improvements in metagenomic virus detection by simple pretreatment methods
AU - Fomsgaard, Anna S.
AU - Rasmussen, Morten
AU - Spiess, Katja
AU - Fomsgaard, Anders
AU - Belsham, Graham J.
AU - Fonager, Jannik
N1 - Publisher Copyright: © 2022 The Authors
PY - 2022
Y1 - 2022
N2 - Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.
AB - Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.
KW - HPV
KW - Metagenomics
KW - Nanopore sequencing
KW - Point of care detection
KW - SARS-CoV-2
U2 - 10.1016/j.jcvp.2022.100120
DO - 10.1016/j.jcvp.2022.100120
M3 - Journal article
C2 - 36945677
AN - SCOPUS:85141826745
VL - 2
JO - Journal of Clinical Virology Plus
JF - Journal of Clinical Virology Plus
SN - 2667-0380
IS - 4
M1 - 100120
ER -
ID: 330900831