In September and October 2001, at least seven envelopes containing significant quantities of B. anthracis spores were mailed through the U.S. postal system to two senators at their congressional offices in the District of Columbia and to media organizations in New York City and Boca Raton, Florida. According to the FBI, the evidence supports the conclusion that the mail attacks occurred on two separate occasions. The two letters of the first attack were postmarked September 18, 2001, and sent to NBC News and the New York Post, both in New York City. Three weeks later, two letters postmarked October 9, 2001, were mailed to two senators—Thomas Daschle and Patrick Leahy—at their Washington, D.C., offices. Other letters were sent to ABC, CBS, and American Media, Inc. Hard evidence of the attacks surfaced on October 3, 2001, when Robert Stevens, an American Media Inc. employee who worked in Boca Raton, Florida, was diagnosed as having contracted inhalational anthrax, from which he later died. However, because a contaminated envelope or package was not recovered in Florida, the agencies could not initially establish how the B. anthracis spores were delivered. According to the Postal Service, the combination of the Florida incident and the opening of the letter to Senator Tom Daschle on October 15 established the link to the U.S. mail system. At least 22 victims contracted anthrax as a result of the mailings. Eleven individuals developed inhalational anthrax, and another 11 developed cutaneous infections. Five of the inhalational anthrax victims died from their infections. The attack highlighted the need for enhanced capabilities for full forensic exploitation and interpretation of microbial evidence from acts of bioterrorism. Ideally, forensic evidence obtained in an investigation is sufficient to support conclusions about the culpability of a group, an individual, or the source of material used in such an act. Forensic evidence is used to support conclusions by classifying evidence into one of several categories that distinguish possible sources from one another. While classification does not unequivocally demonstrate a connection with an individual or a single source, it can be used to reduce the number of possible sources and thus can provide important leads in an investigation. The development and application of microbial forensics was essential to the FBI’s scientific investigation, which relied heavily on genetics and comparative genomics to classify the spore materials used in the attack, reduce the number of possible sources and suspects, and provide investigative leads. In fact, according to the NAS, this investigation accelerated the development of the then nascent field of microbial forensics. The FBI’s investigation, assisted by government, university, and commercial laboratories, was an effort to develop the physical, chemical, genetic, and forensic profiles of the anthrax spores in the letters and envelopes used in the attacks. The investigation employed myriad traditional and novel investigative and scientific methods. The scientific methods involved efforts to develop the physical, chemical, genetic, and forensic profiles of the anthrax spores and letters and envelopes used in the attacks so as to identify the source of the spores. The FBI faced many difficult and complex scientific challenges over the course of this investigation, according to the NAS. New microbial forensic methods were developed and implemented over several years, and some of them provided valuable evidence and significant leads in the case. For example, according to the FBI, new methods to determine the source of the growth media for the mailed spores were inconclusive while the use of the genetic mutations provided an investigative lead. By October 2001, CDC had identified the microorganism used in the attack as Bacillus anthracis (B. anthracis). This was a key step in the classification of the microorganism used in the attack letters and was one of the first scientific findings that allowed the FBI to begin to reduce the number of possible sources of the spores. B. anthracis is a gram positive, rod-shaped bacterium that causes the disease anthrax. It is a member of the larger genus Bacillus that includes other commonly found species, such as B. cereus, B. subtilis, and B. thuringiensis. B. anthracis, a species of Bacillus that can be found on all continents except Antarctica, typically shows little genetic variation among isolates. However, during the investigation scientific methods were being developed that allowed scientists to find some genetic differences among natural isolates of B. anthracis. Applying these methods allowed the FBI to refine the classification of the spores used in the attack and further reduce the number of possible sources of the spores. Even in the most homogeneous species, some differences are usual in genome sequences among populations. Although few in number, these differences are sufficient to characterize subgroups, or “strains.” in the attack letters. In fact, this scientific evidence allowed the FBI to focus its investigation on the limited number of laboratories that had had access to the Ames strain before the attacks (see figure 1). While classifying the spore material as the Ames strain was instrumental in reducing the number of possible sources, it was not sufficient by itself to definitively identify the source of the material used in the attack as a single laboratory, flask, or person. The FBI then sought to identify additional characteristics of the spores used in the attack that could further discriminate between possible sources of the Ames strain. Scientists from the Department of Defense (DOD) tested samples of the spore materials found in the letters and identified several morphological variants (or morphs).the spores from the letters over an extended period observed that a small percentage of the colonies differed in texture, color, and growth patterns A laboratory technician who had grown (cultured) from those typical for the Ames strain of B. anthracis, referred to in figure 2 as the “wild type.” In an effort to identify the source of the letters, investigators and FBI scientists began to evaluate whether they could first identify and characterize these morphs genetically and then determine whether any of them were present in the repository of Ames samples. This involved genome sequencing to identify whether specific deoxyribonucleic acid (DNA) sequences underlay the morphs. Eventually, as shown in figure 2, the morphs were associated with several types of genetic mutations: duplications, single nucleotide polymorphisms (SNP), and deletions, referred to as INDELS. Afterward, over several years, outside contractors’ laboratories developed and validated several genetic tests to analyze the B. anthracis samples for the presence of certain genetic mutations. Specifically, the testing revealed the presence or absence in a sample of a specific DNA sequence (that is, the genetic mutation) associated with a given morph. The FBI contractors generally referred to the tests they developed as A1, A3, D, and E. Commonwealth Biotechnologies (CBI) developed the two A tests (A1 and A3), which targeted two different DNA sequences; and the Institute for Genomic Research (TIGR) developed the E test, targeting another DNA sequence. However, unlike the others, the Illinois Institute of Technology Research Institute (IITRI) and Midwest Research Institute (MRI) both developed a test targeting the same DNA sequence. For clarity, we refer to the IITRI-developed test as D-1 and the MRI- developed tests as D-2. Genetic test A1: detects the presence of a specific duplicated DNA sequence associated with morph A; Genetic test A3: detects a different duplicated DNA sequence from that targeted by A1 but also associated with morph A; Genetic test D-1: detects the presence of a specific deleted DNA sequence associated with morph D; Genetic test D-2: detects the presence of the same deleted DNA sequence associated with morph D as that targeted by the D-1 test; and Genetic test E: detects a deleted DNA sequence associated with morph E. In 2002, the FBI began collecting samples from laboratories in possession of the Ames strain to compare them with the material used in the attack. A grand jury issued subpoenas to 16 domestic laboratories and the FBI requested submissions from 3 foreign laboratories that investigators had determined possessed the Ames strain. The subpoenas required each laboratory to identify and submit two representative samples from each distinct stock of the Ames strain it held. The subpoena included instructions to the laboratories on how to identify, select, and submit samples to the FBI. Laboratories were required to ship sample submissions to DOD scientists at USAMRIID for preparation and entry into the FBI repository of Ames samples. In addition to the samples submitted in response to the subpoena, searches were conducted at three domestic laboratories to ensure that samples were taken from each stock of Ames strain in those facilities. The FBI assembled a repository of 1,070 Ames strain samples, of which From 2004 through 2007, each of the 1,059 viable 1,059 were viable. repository submissions was compared to the evidentiary material using the five genetic tests (see figure 3). The results of the genetic testing indicated that only 8 of the 1,059 FBI repository Ames samples tested positive for the presence of the four genetic mutations originally found in the anthrax letter evidence. Using submission records, investigators concluded that these 8 samples were derived from a single source—a flask identified as RMR-1029. According to the FBI, this information constituted a groundbreaking lead in the development in the investigation. It allowed the investigators to reduce drastically the number of possible suspects, because only very few individuals had ever had access to this specific flask. Armed with this new information obtained from the scientific evidence, the task force focused its investigation on researchers who had had access to the laboratory at USAMRIID where RMR-1029 was stored. In 2008, the FBI sought to conduct statistical analyses in order to determine (1) the probative value of genetic markers found in a sample, and (2) possible inferences regarding the relationships of similar samples. A contractor submitted the Final Statistical Analyses Report in October 2008.