Teresa M. Tiedge* and Kelly Ann Meiklejohn | North Carolina State University Abstract: Soil and dust are often submitted to crime laboratories as trace evidence and can be used to link an individual to a crime scene or to determine an evidentiary sample’s origin. Methodologies that are routinely applied to analyze these geologic materials aim to characterize their physical properties (e.g., color, pH) and inorganic components (e.g., mineral content). However, sample size is often a limiting factor in these analyses; supplemental methods requiring a small amount of geologic material as input could provide additional evidentiary information from evidence. DNA metabarcoding is a commonly used approach to identify the biological taxa present in various environmental samples by amplifying and sequencing short, informative regions of the genome and is not restricted by sample amount. The goal of this research was to determine the utility and stability of environmental DNA (eDNA) from four biological taxa associated with soil and dust for sample-to-sample comparisons and sample origin. In this study, five mock geologic evidence items were collected monthly from agricultural and urban locations in North Carolina over a 1-year period. Mock items included (a) soil removed from t-shirts, boot soles, and trowels, (b) exposed dust collected from brick pavers using polyurethane swabs, and (c) dry dust from air filters (approximately 1” × 1” area used). DNA was isolated from mock geologic evidence using the QIAGEN DNeasy® PowerSoil® Pro Kit, and DNA metabarcoding was applied to characterize bacteria (16S), fungi (ITS1), arthropods (COI), and plants (ITS2, trnL) associated with each sample (n = 1026). Libraries were generated using custom indexed primers and were subsequently sequenced using the Illumina® MiSeq™. Raw sequencing reads were processed through a bioinformatic pipeline that removes primer sequences, identifies amplicon sequence variants (ASVs) via DADA2, and searches the ASVs against GenBank® for taxonomic identification. This presentation will focus on the experimental design and workflow and will include a preliminary assessment of temporal and spatial variables on the recovery of bacteria, fungi, arthropods, and plants from mock geologic evidence.