FBIR samples was deemed an impractical method for several reasons. First, phenotypic screens of the types described above are relatively slow, labor intensive, and highly dependent on the trained eyes of the investigator to identify variant colonies. Second, similar phenotypic variations can be associated with different genetic alterations located in either the same or widely separated genetic loci. The presence of similar colony morphotypes in two samples would not provide direct genetic evidence to link the two sample populations. Third, phenotypic screens are insensitive and do not reliably detect rare variants. Identification of the specific mutations associated with each phenotypic variation was required for the development of definitive assays to detect the presence of shared mutations in multiple strains within the repository. Such DNA-based assays are rapid, sensitive, compatible with high-throughput methods, and definitive to the level of nucleotide sequence.

Scientists selected representative morphotype isolates as well as control wild-type isolates from each of three letter samples for detailed genetic analysis. Several criteria were used for this selection. First, the scientists needed to be able to distinguish the variants from the wild-type colonies on plates. Second, these particular morphotypes must have been present at a high enough frequency for the scientists to identify them repeatedly. The third essential criterion was the apparent presence of the morphotype in each of the three letter samples (Leahy, Daschle, New York Post) that were subjected to this analysis. The final selection of morphotypes focused on four variants: A, B, C/D, and E. There were other morphotypes found in the letter materials, but they were not used for further forensic testing. Worsham and colleagues at USAMRIID quantified the percentages of variants by randomly picking about 370 isolated colonies from plates made using dilutions from the Leahy letter. These colonies were 79 percent wild-type morphology, 6.7 percent C/D morphotype, 1.1 percent B morphotype, 1.3 percent A morphotype, and 4.9 percent E morphotype (other morphotypes accounted for the remaining fraction) (FBI Documents, B1M2D12). It is important to note that two identical-looking morphotypes need not, and often did not, have the same genotype. Indeed, as discussed in the next section, two independent isolates exhibiting similar colony morphotypes might have mutations in different genes or even different mutations in the same gene. Also, some colonies identified as morphotypic variants may not have had any mutation, as the distinction between genetic and nongenetic variation is not always clear. Thus, it was crucial to identify differences in nucleotide sequence as an unambiguous signature of different mutant subpopulations.

5.5.5 Whole Genome Sequencing of Morphotype Isolates

To determine whether the genetic alterations associated with each colony morphotype might be suitable for use as forensic markers, the genome sequences of multiple morphotype isolates were determined (Table 5-2). Genomic DNA



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