Evaluation of 16S rRNA Hypervariable Regions for Bioweapon Species Detection by Massively Parallel Sequencing

Evaluation of 16S rRNA Hypervariable Regions for Bioweapon Species Detection by Massively Parallel Sequencing

Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parall...

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Published in:International journal of microbiology Vol. 2020; no. 2020; pp. 1 - 11
Main Authors: Silva, Rosane, Budowle, Bruce, Woerner, August E., Cabral, Bianca C. A., Azevedo-Martins, Allan C., Gomes, Priscila da S. F. C., Dias, Victor H. G., Moura-Neto, Rodrigo S.
Format: Journal Article
Language:English
Published: Cairo, Egypt Hindawi Publishing Corporation 26-09-2020
Hindawi
John Wiley & Sons, Inc
Hindawi Limited
Subjects:
Bacteria
Biohazards
Biological weapons
Deoxyribonucleic acid
DNA
DNA sequencing
E coli
Forensic science
Gene amplification
Genes
Genomes
Genomics
International economic relations
Medical research
Microbiology
Microorganisms
Multiprocessing
Nucleotide sequencing
Pathogens
Phylogenetics
Phylogeny
RNA
rRNA 16S
Salmonella
Scientific equipment and supplies industry
Sensitivity enhancement
Software
Species
Strains (organisms)
Brazil
United States
Germany
United States > US
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Summary:Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parallel sequencing (MPS) offers enhanced sensitivity and specificity for microbiological analyses. In addition, 16S rRNA target amplification followed by MPS facilitates the combined use of multiple markers/regions, better discrimination of sample background, and higher sample throughput. We designed a novel set of 16S rRNA gene primers for detection of bacterial species associated with clinical, bioweapon, and biohazards microorganisms via alignment of 364 sequences representing 19 bacterial species and strains relevant to medical and forensics applications. In silico results indicated that the hypervariable regions (V1V2), (V4V5), and (V6V7V8) support the resolution of a selected group of bacteria. Interspecies and intraspecies comparisons showed 74.23%–85.51% and 94.48%–99.98% sequencing variation among species and strains, respectively. Sequence reads from a simulated scenario of bacterial species mapped to each of the three hypervariable regions of the respective species with different affinities. The minimum limit of detection was achieved using two different MPS platforms. This protocol can be used to detect or monitor as low as 2,000 genome equivalents of bacterial species associated with clinical, bioweapon, and biohazard microorganisms and potentially can distinguish natural outbreaks of pathogenic microorganisms from those occurring by intentional release.
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Academic Editor: Todd R. Callaway
ISSN:1687-918X
1687-9198
DOI:10.1155/2020/8865520