Assessing three soil removal methods for environmental DNA analysis of mock forensic geology evidence.

Soil is useful in criminal investigations as it is highly variable and readily transferred. Forensic geologists use several different techniques to removal soil from evidence prior to the analysis of inorganic components. There has been recent interest from the forensic science community to analyze environmental deoxyribonucleic acid (eDNA) associated with soil to augment existing forensic analyses. Notably however, limited research has been conducted to compare commonly used soil removal methods for downstream eDNA analysis. In this study, three soil removal methods were assessed: picking/scraping, sonication, and swabbing. Three mock evidence types (t-shirts, boot soles, and trowels) were sampled in triplicate with each removal method (n = 27). Soil samples underwent DNA isolation, quantification, and amplification of four genomic barcode regions: 16S for bacteria, ITS1 for fungi, ITS2 for plants, and COI for arthropods. Amplicons were prepared into libraries for DNA sequencing on an Illumina MiniSeq. DNA concentrations were highest in picked/scraped samples and were statistically significant compared with swabbed and sonicated samples. Amplicon sequence variants (ASVs) were identified, and removal methods had no impact on the recovery of the total number of target ASVs. Additionally, when assessing each sample in multidimensional space, picked/scraped samples tended to cluster separately from swabbed and sonicated samples. The soil core used a reference in this study also clustered with the picked/scraped samples, indicating that these samples may be more reflective of the communities collected from soil cores. Based on these data, we identified that picking/scraping is an acceptable soil removal method for eDNA analysis.