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Then there is the issue of the inverted plasmid.

Bioforensics Program BAA08-04
Bacterial Population Genetics in a Forensics Context
(BPGFC)
Published: 03/04/2008
Amended: 4/16/2008

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The threat of terrorist or criminal use of pathogenic organisms and their toxins remains of great concern in the United States. There are vulnerabilities and needs to perform microbial forensic analyses for attribution purposes in a rigorous scientific manner. As part of the effort to deter biological terrorism and strengthen the law enforcement response to such an
act, Homeland Security Presidential Directive (HSPD) 10, “Biodefense for the 21st Century” established a dedicated central microbial forensic laboratory known as the National Bioforensics Analysis Center (NBFAC), as part of the Department of Homeland Security to provide bioforensics analysis of evidence associated with the event. The NBFAC operates in partnership with the Federal Bureau of Investigation (FBI), the lead investigative agency in acts of terrorism. This BAA seeks research in the discipline of bacterial population genetics to support the NBFAC’s mission for evidentiary analysis and
interpretation of results to support the FBI’s criminal investigation. The ultimate goal of this joint mission is to provide sound scientific data and analysis supporting attribution analysis leading to the capture, indictment, and prosecution of the perpetrator(s) of the biocrime or terrorist attack.

The NBFAC has instituted a robust molecular biology program to enhance the genomic analysis of the major biological threat agents. The NBFAC continues to expand its operational capabilities for the identification and characterization of the major CDC category A and B agents. The Bioforensics Research and Development Program continues research investments in next generation technologies to support molecular biology and
genotyping techniques capable of characterizing a biothreat agent at the isolate level to better understand the microorganism’s origin and evolutionary history. However, a significant gap exists in the forensics communities understanding of the meaningful interpretation of genotyping/profiling results. Unlike its applications in human DNA analysis, which deals with one species exhibiting diploid sexual reproduction, the bacterial
threat agents pose a more complex problem for the interpretation of genomic data. The bacterial threat agents are haploid organisms propagating asexually and have always been thought to be purely clonal in nature, but population variation does exist even within what have been assumed to be clonal populations, such as a single colony on a Petri-plate. In addition, there are other substantial genetic issues related to interpretation including horizontal gene transfer, recombination, gene duplication and deletion. Hence, there is need for a better understanding of the underlying genomic and population dynamics at both intra and inter-species levels.
Program Objectives
The goal of this research program is to develop algorithms and/or analytical tools that will assist biocrime and bioterror investigations by providing precision and statistical power to inferences concerning the degree of relatedness (based upon matching comparisons) of biothreat agents. The focus of this effort will be on research in the areas of microbial
ecology, population dynamics, genetic stability, host-pathogen interaction, statistical inference, and match criteria for the following select agents: Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Francisella tularensis, Brucella spp., Escherichia coli O157:H7, Burkholderia mallei, Burkholderia pseudomallei. The objectives include:

¥ Improving our understanding of the population genetics of these pathogens to include research on genome stability, host preferences and interactions, genetic mobility of virulence factors, identification of polymorphic sites and mutational hot spots, geographical distribution, microbial ecology, pathogen lifecycles, natural reservoirs and effects of host – pathogen interaction.
¥ Improving methods for determining rates of mutation and recombination of the pathogen genomes and the identification of adaptive mutations that can have forensic utility
¥ Establishing match criteria for discriminating “difference” or “sameness” in
sample comparisons. Define the parameters for inclusion or exclusion in the
context of a known sample and questioned sample. This must be backed by
statistical parameters for acceptance or rejection of the null hypothesis
¥ Developing efficient, economical, and statistically rigorous sampling strategies to acquire spatially referenced genetic information on reservoirs of these pathogens
¥ Developing bioinformatics-based analytical tools for supporting hypotheses
testing regarding pathogen origin that go beyond current phylogeny-based
inferential methods and can meet forensic (legal) admissibility requirements of relevance and reliability.

Background and Motivation
Current forensic analysis of biological threat agents is impeded by lack of knowledge of the underlying population genetics and ecology of the pathogens. In addition, there is a void in the availability of bioinformatics tools necessary to analyze and validate the data that forensics investigators collect from incidence sites. An understanding of pathogen population genetics including ecology, phylogeny, lifecycles, genome stability, mutation rates, recombination rates, epidemiology, host preferences and interactions, geographic distribution and other source information, virulence factors, polymorphic sites and mutation hot spots will be necessary to interpret the results from the application of multiple typing methods (e.g. MLST, VNTR, SNP, InDel, SSRs etc) and use of these techniques to compare “likeness” of isolates associated with a bioforensic investigation. The population genetics will likely be different for each pathogen; hence the typing strategy is also likely to be different. For example, pathogens with stable genomes might be more amenable to source tracking through SNP analysis than pathogens with unstable genomes. Furthermore, as typing data on the pathogens is generated, it will need to be organized, archived and managed in a form that is functional and accessible for forensic analysis. To further complicate the genotyping challenge is the rapid emergence of Next-Generation
whole genome sequencing technologies, e.g., Roche (GS-20 FLX/454 Pyrosequencing), Illumina (Genome Analyzer System), and ABI (SOLiD) which will provide faster and cheaper whole genome sequences. These platforms will potentially displace many of the traditional genotyping techniques in the near future by providing a much richer body of data associated with a whole genome sequence. This will increase by orders of magnitude the sequence data available for microorganisms. Therefore, development of statistically based methodologies to compare whole genome sequences and sort through the maze of data for match comparisons is critical to adequately make use of these powerful new methods.
In addition to an understanding of the population genetics of the pathogens, bioinformatic tools that can bring statistical power and degrees of confidence to the results will need to be developed in order to make an assessment of “match” criteria for comparative forensic purposes. In other words, can reliable inferences be made when a biological threat agent
found at a crime scene is compared to a sample found at a suspect’s home laboratory or place of employment? These tools should have the power to effectively describe the quantitative criteria and provide statistical support as to the likelihood or degree of similarity. These computational tools will need to be developed, standardized and well documented and will be required to produce understandable, explainable and defensible results in a forensic setting.

The program will be separated into two phases. The phase I program goal is to produce a paper study that not only reviews the current level of understanding in terms of population genetics and bioinformatics tools available for each of the eight agents, but also makes recommendations for follow on experiments and research plan that can be performed in
phase II to fill in the knowledge gaps necessary for rigorous forensic data analysis. Phase I awardees have the opportunity to apply for phase II. The phase II program goal is to develop and perform experimental studies that fill in the gaps in current knowledge of the population genetics of the eight select agents as well as to develop new algorithms and
bioinformatics tools to enhance statistical analysis and inference for forensic pathogen analysis. Phase II will consist of two tasks: a computational/bioinformatics task and a laboratory-based experimental task. Proposers may choose to perform one or both tasks or establish a team with other institutions to address both tasks. Teaming is highly encouraged.

Comment:

For the identification of those responsible for the Fall 2001 mailings, researchers are encouraged to go to “Summons to Conquest.”

http://www.anthraxandalqaeda.com

“Does it look like I’m negotiating?” — George Clooney, in Michael Clayton