Targeted amplification for enhanced detection of biothreat agents by next-generation sequencing
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Global Security Program. Bioinformatics
- Naval Medical Research Center, Fort Detrick, MD (United States); Henry M. Jackson Foundation, Bethesda, MD (United States)
- Thermo Fisher Scientific, South San Francisco, CA (United States)
- Naval Medical Research Center, Fort Detrick, MD (United States)
Background: Historically, identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. However, these methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Thus, there has been a need for assays that do not suffer as greatly from these limitations and/or biases. Recent advances in library preparation technologies for Next-Generation Sequencing (NGS) are focusing on the use of targeted amplification and targeted enrichment/ capture to ensure that the most highly discriminating regions of the genomes of known targets (organism-unique regions and/or regions containing functionally important genes or phylogenetically-discriminating SNPs) will be sequenced, regardless of the complex sample background. Results: In the present study, we have assessed the feasibility of targeted sequence enhancement via amplification to facilitate detection of a bacterial pathogen present in low copy numbers in a background of human genomic material. Our results indicate that the targeted amplification of signature regions can effectively identify pathogen genomic material present in as little as 10 copies per ml in a complex sample. Importantly, the correct species and strain calls could be made in amplified samples, while this was not possible in unamplified samples. Conclusions: The results presented here demonstrate the efficacy of a targeted amplification approach to biothreat detection, using multiple highly-discriminative amplicons per biothreat organism that provide redundancy in case of variation in some primer regions. Importantly, strain level discrimination was possible at levels of 10 genome equivalents. Similar results could be obtained through use of panels focused on the identification of amplicons targeted for specific genes or SNPs instead of, or in addition to, those targeted for specific organisms (ongoing gene-targeting work to be reported later). Note that without some form of targeted enhancement, the enormous background present in complex clinical and environmental samples makes it highly unlikely that sufficient coverage of key pathogen(s) present in the sample will be achieved with current NGS technology to guarantee that the most highly discriminating regions will be sequenced.
- Research Organization:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- Grant/Contract Number:
- AC52-07NA27344
- OSTI ID:
- 1629043
- Journal Information:
- BMC Research Notes, Vol. 8, Issue 1; ISSN 1756-0500
- Publisher:
- BioMed CentralCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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