Random Whole Metagenomic Sequencing for Forensic Discrimination of Soils

Abstract

Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.

Bioinformatics Approach to Assess the Biogeographical Patterns of Soil Communities: The Utility for Soil Provenance,

“…Forensic soil analyses are usually conducted by comparing questioned samples with those of known origin to evaluate whether they are similar (inclusion) or different (exclusion) based on physical characteristics (e.g., soil color, texture, consistency, density, porosity, and particle size) (2)(3)(4) and chemical properties (i.e., mineralogy and elemental composition) (1,(4)(5)(6). With the growth of molecular techniques, research has shown the potential of soil DNA profiling as a reliable method for forensic soil analyses (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The current ecological hypothesis states that the soil type (e.g., chemical and physical properties) determines which microbes and plants occupy a particular soil (20)(21)(22) and provides the foundation for soil provenance studies to assist in intelligence gathering or forensic applications.…”

“…Studies have shown the potential and effectiveness of using microbial DNA from soil to identify the origin of a soil sample (7)(8)(9)11,14,(16)(17)(18). A majority of the studies explore the bacterial 16S rRNA genes using terminal restriction length fragment polymorphism (T-RFLP) (7,9,10) or length heterogeneity polymerase chain reaction (LH-PCR) (8,23).…”

The Future of Forensic Biology

“…The microbes and hence the microbial DNA differs in males and females and therefore after sex, the microbes in both the male and the female appear to swap, indicating that a sexual act occurred between a particular man and woman. The microbial forensics has been broadened by metagenomic analysis of the microbes from the surroundings also such as from soil [37] in case of bioterroism/murderous crimes, which may also help in collecting and corroborating evidences to correlate with the incident.…”

A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils

“…Current efforts that have used molecular-based approaches such as DNA barcoding to document the biodiversity within a soil sample have primarily been focused on a bulk metagenomic approach [41][42][43][44][45][46][47][48][49][50]. Using conserved primers for the desired barcode regions, individual taxa can be amplified and sequenced simultaneously (i.e., massively parallel sequencing) from a single bulk soil extraction.…”