Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents.
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 fromin 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.IMPORTANCEBoth 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.
Department of Neurology, University of California, San Francisco, San Francisco, California, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.
Michael R
MR
Wilson
Integrative Program in Quantitative Biology, Bioinformatics, University of California, San Francisco, San Francisco, California, USA.
Greg
0000-0002-3448-3819
G
Fedewa
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA.
Mark D
MD
Stenglein
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
Judith
J
Olejnik
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
Linda J
LJ
Rennick
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
Sham
S
Nambulli
Rocky Mountain Veterinary Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
Friederike
F
Feldmann
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
W Paul
WP
Duprex
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
John H
0000-0002-8867-7256
JH
Connor
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
Elke
E
Mühlberger
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
Joseph L
JL
DeRisi
metagenomics; pathogen tracking; phylogenetic analysis