Soil is a common form of trace evidence, although it usually qualifies only as class evidence. Microbiological methods have been used to assess if the microbial makeup, primarily bacterial, of soil might be valuable for forensic identification. However, a shortcoming of previous studies is that microbial DNA was assayed shortly after soil collection or the soils were frozen until processing, which is not a realistic forensic scenario. The bacterial makeup of soil is transient and may be influenced by the length of time soil has been removed from a habitat, the storage temperature of known soils for comparison, and the amount of soil recovered from evidence. In this research, next-generation sequencing was used to generate bacterial profiles from diverse habitat soils that were collected from various evidence items over time, stored at four different temperatures, and processed at different masses. Bacterial abundance charts and nonmetric multidimensional scaling plots provided visual representations of the bacterial profiles, and two supervised classification techniques were used to statistically analyze them. Over time, the bacterial composition of the soil evidence displayed specific, consistent changes, and the soil evidence profiles grouped with, although drifted away from, the habitat of origin in multidimensional space. Storing known soils under the same conditions as the aged soil evidence improved the associations between them. Finally, all soil masses processed, including trace amounts, correctly assigned to the habitat of origin. Ultimately, our understanding of the bacterial changes will allow bacterial profiling of soil evidence to be more individualizing and strengthen the potential associations of a suspect, victim, or evidence item with a crime scene.
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