Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents

ABSTRACT Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identi...

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Autores principales:	Michael R. Wilson, Greg Fedewa, Mark D. Stenglein, Judith Olejnik, Linda J. Rennick, Sham Nambulli, Friederike Feldmann, W. Paul Duprex, John H. Connor, Elke Mühlberger, Joseph L. DeRisi
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	metagenomics pathogen tracking phylogenetic analysis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/2566511182bd48c2bca99e1a3a8b7567
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spelling	oai:doaj.org-article:2566511182bd48c2bca99e1a3a8b75672021-12-02T19:48:49ZMultiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents10.1128/mSystems.00058-162379-5077https://doaj.org/article/2566511182bd48c2bca99e1a3a8b75672016-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00058-16https://doaj.org/toc/2379-5077ABSTRACT Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identity, the extent of genomic variation, and the presence of contaminants. Double-stranded cDNA MDS libraries were constructed from RNA extracted from in vitro-passaged stocks of six viruses (La Crosse virus, Ebola virus, canine distemper virus, measles virus, human respiratory syncytial virus, and vesicular stomatitis virus). Each library was dual indexed and pooled for sequencing. A custom bioinformatics pipeline determined the organisms present in each sample in a blinded fashion. Single nucleotide variant (SNV) analysis identified viral isolates. We confirmed that (i) each sample contained the expected microbe, (ii) dual indexing of the samples minimized false assignments of individual sequences, (iii) multiple viral and bacterial contaminants were present, and (iv) SNV analysis of the viral genomes allowed precise identification of the viral isolates. MDS can be multiplexed to allow simultaneous and unbiased interrogation of mixed microbial cultures and (i) confirm pathogen identity, (ii) characterize the extent of genomic variation, (iii) confirm the cell line used for virus propagation, and (iv) assess for contaminating microbes. These assessments ensure the true composition of these high-priority reagents and generate a comprehensive database of microbial genomes studied in each facility. MDS can serve as an integral part of a pathogen-tracking program which in turn will enhance sample security and increase experimental rigor and precision. IMPORTANCE Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock.Michael R. WilsonGreg FedewaMark D. StengleinJudith OlejnikLinda J. RennickSham NambulliFriederike FeldmannW. Paul DuprexJohn H. ConnorElke MühlbergerJoseph L. DeRisiAmerican Society for Microbiologyarticlemetagenomicspathogen trackingphylogenetic analysisMicrobiologyQR1-502ENmSystems, Vol 1, Iss 4 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	metagenomics pathogen tracking phylogenetic analysis Microbiology QR1-502
spellingShingle	metagenomics pathogen tracking phylogenetic analysis Microbiology QR1-502 Michael R. Wilson Greg Fedewa Mark D. Stenglein Judith Olejnik Linda J. Rennick Sham Nambulli Friederike Feldmann W. Paul Duprex John H. Connor Elke Mühlberger Joseph L. DeRisi Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
description	ABSTRACT Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identity, the extent of genomic variation, and the presence of contaminants. Double-stranded cDNA MDS libraries were constructed from RNA extracted from in vitro-passaged stocks of six viruses (La Crosse virus, Ebola virus, canine distemper virus, measles virus, human respiratory syncytial virus, and vesicular stomatitis virus). Each library was dual indexed and pooled for sequencing. A custom bioinformatics pipeline determined the organisms present in each sample in a blinded fashion. Single nucleotide variant (SNV) analysis identified viral isolates. We confirmed that (i) each sample contained the expected microbe, (ii) dual indexing of the samples minimized false assignments of individual sequences, (iii) multiple viral and bacterial contaminants were present, and (iv) SNV analysis of the viral genomes allowed precise identification of the viral isolates. MDS can be multiplexed to allow simultaneous and unbiased interrogation of mixed microbial cultures and (i) confirm pathogen identity, (ii) characterize the extent of genomic variation, (iii) confirm the cell line used for virus propagation, and (iv) assess for contaminating microbes. These assessments ensure the true composition of these high-priority reagents and generate a comprehensive database of microbial genomes studied in each facility. MDS can serve as an integral part of a pathogen-tracking program which in turn will enhance sample security and increase experimental rigor and precision. IMPORTANCE Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock.
format	article
author	Michael R. Wilson Greg Fedewa Mark D. Stenglein Judith Olejnik Linda J. Rennick Sham Nambulli Friederike Feldmann W. Paul Duprex John H. Connor Elke Mühlberger Joseph L. DeRisi
author_facet	Michael R. Wilson Greg Fedewa Mark D. Stenglein Judith Olejnik Linda J. Rennick Sham Nambulli Friederike Feldmann W. Paul Duprex John H. Connor Elke Mühlberger Joseph L. DeRisi
author_sort	Michael R. Wilson
title	Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
title_short	Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
title_full	Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
title_fullStr	Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
title_full_unstemmed	Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents
title_sort	multiplexed metagenomic deep sequencing to analyze the composition of high-priority pathogen reagents
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/2566511182bd48c2bca99e1a3a8b7567
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Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents

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