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Abstract
High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.
Highlights
More than a decade ago the United States experienced a simple but effective biological attack in which Bacillus anthracis endospores were placed in envelopes and delivered by the US postal service to intended victims [1,2,3,4]
The limitations of methods used in microbial forensics to generate results must be reliable and defensible and the process(es) of validation will contribute substantially in defining confidence associated with an assay, method, or system
The High throughput sequencing (HTS) process was described in some detail so that analysts, who are not experienced researchers, will have guidance on the features and criteria that should be addressed during a validation
Summary
Microbial forensics involves analysis of microbe-related materials found at a crime scene, suspected laboratory, and so on, for forensic attribution and, can be pivotal for developing investigative leads. High throughput sequencing (HTS) vastly improves the possibility that the forensic and scientific communities will be able to assign features to bio-forensic evidence, such as specific identity for unknown or emerging pathogens, sample or microbe origin, antibiotic sensitivity, evidence of genetic engineering and virulence profile. That a number of laboratories can afford HTS systems, community-accepted validation guidelines or standards are needed. As with any analytical tool(s) for forensic application, the utility of HTS operating conditions and tolerances and interpretation guidelines must be carefully defined. We define the criteria and offer a process for validation of HTS systems in microbial forensics. Higher level of power and analytical value and, greater protection for the public and the nation’s safety and security
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