Forensic & Investigative Sciences Program Theses and Dissertations

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High-throughput Amplicon Sequencing Optimization for Downstream Prediction Purposes
(2025-08) Dopkins, Nichole; Walsh, Susan; Picard, Christine; Breslin, Krystal
Alongside short tandem repeat (STR) typing, forensic DNA phenotyping (FDP) can enhance forensic DNA analysis by inferring externally visible characteristics and biogeographical ancestry from single-nucleotide polymorphisms (SNPs). This project involves the optimization of a high-throughput amplicon sequencing assay targeting ~2,000 SNPs associated with pigmentation, facial morphology, and ancestry. Over 400 samples were processed through a genotyping pipeline involving DNA extraction, library preparation with a modified version of a commerical FDP assay, MiSeq FGx sequencing, genotype calling, and quality and ancestry inference assessments. The substantial dataset produced with this project will supply downstream applications in pigmentation and facial morphology prediction modeling. This work also advances FDP toward wide-scale integration into forensic laboratories and demonstrates how SNP-based genotyping can extend investigative leads when traditional STR matching is insufficient.
Detection of Insensitive Munitions Using Blow Flies as Environmental Sampling Devices
(2025-08) Zieminski, Alexis Theresa; Manicke, Nicholas; Goodpaster, John; Fedick, Patrick
Insensitive munitions are high explosives that are less shock sensitive than historical high explosives. Munitions compounds 2,4-dinitroanisole, nitrotriazolone, and nitroguanidine commonly comprise the explosives. As the demand for insensitive munitions has increased, contamination from manufacturing sites and training ranges has also increased. Munitions compounds can seep out of soil into waterways, potentially impacting the environment and contaminating food and water supplies. Therefore, determining the presence of these compounds in the environment is important. Direct sampling by collecting soil and water samples can be difficult due to the need to collect samples over wide areas, sometimes in dangerous, remote, or access restricted areas. In this project, blow flies were used as environmental sampling tools to determine the presence of insensitive munitions in soil. In all fly experiments, blow flies were exposed to an insensitive munitions water-soil mixture. Three types of experiments were conducted: pooling multiple flies during extraction, feeding flies a protein-rich food source prior to munitions, and determining the persistence of munitions in blow flies after a one-time exposure. The amount of munitions present in flies was detected via high performance liquid chromatography-mass spectrometry. Reverse-phase and hydrophilic interaction liquid chromatography (HILIC) methods were developed to detect insensitive munitions. The reverse-phase method detected munitions in soil and blow flies immediately after exposure. However, ion suppression of compounds nitrotriazolone and nitroguanidine by glucose metabolites in aged flies limited the ability for detection with this method. A comparison of single blow fly and multi-blow fly extracts was done. Multi-fly extracts had increased signal to noise ratios, indicating method sensitivity improvement. The development of a HILIC method improved retention of the analytes impacted by ion suppression in reverse-phase liquid chromatography. However, separation of 2,4-dinitroanisole and its primary transformation product 2,4-dinitrophenol was not seen with this method. The HILIC method was used to determine persistence of IM in blow flies after controlled exposures to spiked soil. Blow flies were sampled at 0-, 1-, 3-, and 5-days post exposure at varying temperatures. 2,4-dinitroanisole/2,4-dinitrophenol was detected in flies one day post-exposure. Nitrotriazolone was detected in flies 3- days post feed. Nitroguanidine was detected 5 days post-exposure. This proof-of-concept project proved the ability to detect insensitive munitions in blow flies. Future directions of blow fly analysis include sampling blow flies baited from real-world contamination sites and development of a QuEChERS technique for improved detection from fly matrices.
Utilizing Phormia Regina as an Environmental Sensor for Resource Identification and Biodiversity Monitoring
(2024-08) Jensen, Katharine Theresa; Picard, Christine; Manicke, Nicholas; Dembinski, Gina
Blow flies are a family of carrion insects that are among the first to arrive in the decomposition process. Blow flies are known to ingest carrion, feces, water, and occasionally nectar to meet nutritional requirements. These behaviors make blow flies a unique organism potentially containing genetic material from a variety of sources within one environment. Their global distribution and ease of capture makes them a strong candidate for resource monitoring and identification. While previous studies have evaluated the suitability of blow flies for vertebrate biodiversity estimates, no work has been done looking at their ability to ingest and store genetic material from plants and microbes present in water. It is also not known how long these DNA signals persist in the gut. Through DNA analysis of the blow fly gut, researchers can identify vertebrates that have recently died in an environment, what plant species are present, and what water source the insect utilized. Through lab colony (Phormia regina) feeding experiments, it was determined that at 25 ˚C and 50 % relative humidity, vertebrate and plant DNA persist in the gut for over 120 hours post-ingestion. Wild sample analysis of flies collected from Yellowstone National Park was performed to identify plant species ingested by P. regina in the wild. Following Sanger sequencing, top hits on BLASTn included Brassicales, Juglans cathyensis, and uncultured Candida. This is the first application of environmental DNA analysis techniques to insects for the purpose of plant identification. This work also attempts to characterize microbial profiles of the gut of P. regina for the purpose of water resource identification. Over a two-month collection period, samples were collected from different water resources across Indianapolis. Flies were exposed to these samples in a controlled feeding experiment, followed by sampling at 0- and 72 hours post-exposure. Gut samples were sequenced using Illumina and Operational Taxonomic Unit clustering grouped reads by sequence similarity for identification. Bacteria classes identified included Gammaproteobacteria, Bacteroidia, Flavobacteria, Alphaproteobacteria, Bacilli, Clostridia, Actinobacteria, Betaproteobacteria, and Fusobacteria. Many bacteria classes were common across water samples, although the abundance of each class changed between samples and across time. These unique microbial profiles can be used to identify water resources for potential contamination and chemical dumping. Further work is necessary to generate microbial profiles from the original water sources themselves and for generation of alpha and beta diversities. Overall, this work spans multiple fields. Species identification is important for biodiversity monitoring and environmental surveys. Utilizing blow fly derived DNA allows for detection of living and deceased vertebrates in an environment, plant life, and water quality within one sample. This work also has implications in forensic science, specifically wildlife forensics and chemical detection of clandestine laboratories and chemical weapon compounds.
Characterization of Simple Sequence Repeats in Phormia Regina Miegen (Diptera: Callphoridae)
(2024-08) Waletzko, Cassandra; Picard, Christine; Walsh, Susan; Owings, Charity
Phormia regina Meigen is a forensically relevant species of blow fly, common in North America and used to estimate the minimum postmortem interval in forensic casework. It is also possible to use blow flies to survey the environment for biotic and abiotic information drawn from both larval and adult stages. There are both forensic and environmental uses for genetic analysis of blow flies. Blow fly kinship is especially useful for detecting postmortem movement of a corpse or to assess abundance of carrion in a given location. To test genetic relationships between individuals, discriminatory loci such as microsatellites, or polymorphic tandemly repeated sequences of DNA are necessary. Here, we characterize novel microsatellites generated from the genome of P. regina. Thirty-four candidate polymorphic loci with conserved flanking regions, have been isolated. To date, seven are heterozygous and polymorphic testing in two lab populations and one wild population. The simple sequence repeats characterized here complement existing loci (N = 6) for greater discrimination for testing relationships between individual flies.
Screening and Quantitation of Volatiles from Explosive Initiators and Plastic Bonded Explosives (PBX)
(2024-05) Hecker, Alexis; Goodpaster, John; Manicke, Nick; Beymer, Christina
The detection of explosives and explosive devices based upon the volatile compounds they emit is a long-standing tool for law enforcement and physical security. Towards that end, solid phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) has become a crucial analytical tool for the identification of volatiles emitted by explosives. Previous SPME studies have identified many volatile compounds emitted by common explosive formulations that serve as the main charge in explosive devices. However, limited research has been conducted on initiators like fuses, detonating cords, and boosters. In this study, a variety of SPME fiber coatings (i.e., polydimethylsiloxane (PDMS), polydimethylsiloxane/divinylbenzene (PDMS/DVB), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), carboxen/polydimethylsiloxane (CAR/PDMS), and polyacrylate (PA)) were employed for the extraction and analysis of volatiles from Composition C-4 (cyclohexanone, 2-ethyl-1-hexanol, and 2,3-dimethyl-2,3-dinitrobutane (DMNB)) and Red Dot double-base smokeless powder (nitroglycerine, phenylamine). The results revealed that a PDMS/DVB fiber was optimal. Then, an assortment of explosive items (i.e., detonation cord, safety fuse, slip-on booster, and shape charge) were analyzed with a PDMS/DVB fiber. A variety of volatile compounds were identified, including plasticizers (tributyl acetyl citrate, N-butylbenzenesulfonamide), taggants (DMNB), and degradation products (2-ethyl-1-hexanol). Taggants, like DMNB, are one of the pivotal components added to explosives. These distinctive chemical markers, deliberately introduced during manufacturing to facilitate the identification of explosives, are commonly detected using SPME GC- MS, but their quantitation remains underexplored. To address this, we investigated total vaporization headspace (TV- HS) GC- MS for quantifying taggants in the headspace of Composition C4. Factors effecting the extraction of DMNB, such as shape and age of the sample, and surface depletion, were also examined. The results revealed that the shape of the sample did not affect the amount of DMNB in the headspace but the older the sample, the more DMNB was detected in the headspace. Surface depletion was also seen in samples that were exposed to air for more than one week. Then calibration curves with calibrants of DMNB in acetone were established for quantitation. The average concentration of DMNB in the headspace was determined to be 125 parts per million (ppm).
Paper Spray-Mass Spectrometry Coupled with Pressure Sensitive Adhesive-Based Collection for the Recovery and Detection of Drugs of Abuse
(2023-08) Prunty, Sarah G.; Manicke, Nicholas; Goodpaster, John; McKenna, Josiah
Illicit drug abuse is a widespread issue in the United States and worldwide. Many methods seek to ease the analytical workload required to collect, analyze, and identify these drugs. Paper spray-mass spectrometry (PS-MS) is one response to this analytical workload as it offers a rapid, affordable, and simple means for drug identification by mass spectrometry. This work centers on the use of pressure-sensitive adhesive (PSA) lined paper as a PS-MS substrate for drug recovery and detection. The use of PSA paper as a sampling and analysis substrate has been previously established but is expanded herein with new capabilities and applications. Chapter 2 introduces the combination of color tests followed by PS-MS for presumptive and confirmatory drug identification. Three color tests (cobalt thiocyanate, Simon, or Marquis) were performed on the PSA paper with subsequent drug confirmation occurring by PS-MS. Chapter 3 examines the use of PSA paper and PS-MS for the recovery and detection of fentanyl, fentanyl precursors, and analogs from shipping-related surfaces and in the presence of high amounts of cutting agents. The use of a cartridge that accommodates a full-sized PSA paper ticket was also explored for drug detection. Chapter 4 assesses PS-MS with PSA paper on portable MS instrumentation. Analyte recovery and carryover as well as instrument robustness were evaluated. The color test and PS-MS protocol examined in Chapter 2 was also successfully applied to a portable MS instrument. Application of PS-MS to the portable system highlights the potential fieldability of the technique.
Electrochemical Characterization of Common Cutting Agents Found in Illicit Drugs
(2023-08) Bloom, George; Deiss, Frederique; Manicke, Nicholas; Rainey-Beymer, Christina
Nationwide use of illicit drugs has continued to rise over the last few decades, with more than a two-fold increase in global seizures from 2016 and 2020. Most seized drug samples are complex mixtures of drugs and cutting agents, which can complicate the detection and quantification of the illicit drugs in the sample. The presence of these cutting agents can however be beneficial for source tracing purposes, as the majority of cutting agents are selected based on availability in the area where the bulk drug was prepared. The goal of this work was to conduct a systematic study of the electrochemical characteristics of the most common cutting agents found in illicit drugs using unmodified, commercially available glassy carbon electrodes. The long-term goal is to establish an extensive database of electrochemical characterizations of cutting agents and illicit drugs encountered by law enforcement using unmodified, commercially available electrodes to help expand the developing field of forensic electrochemical analyses. This database could then be referenced for the identification of unknown samples to determine the presence of possible illicit drugs and cutting agents that are present to help guide the analyst in further testing. The standard methods for drug detection include a combination of laboratory testing and field-deployable assays ranging from colorimetric tests to gas chromatography-mass spectrometry instrumentation. These detection methods, as well as relevant literature were investigated in Chapter 1. The most used screening methods for illicit drugs are colorimetric tests; however, these assays are prone to false positives. Chapter 1 introduces the existing applications and current research efforts in forensic electrochemistry by describing relevant electrochemical sensors and methods and examining in particular their performance regarding accuracy, sensitivity, and low-cost claims. This overview highlights the broad possibilities of electrochemical analysis in forensics as well as the opportunities when applied to detection and quantification of illicit drugs, demonstrating the current needs for more systematic and consistent characterizations of cutting agents found in seized-drug samples. Chapter 2 details the material, reagents, and experimental conditions, showing their simplicity, and the standard electrochemical and preparative equipment used geared towards an easy implementation in any analytical laboratory. Chapter 3 describes the systematic voltametric characterizations performed on thirteen common cutting agents: phenacetin hydrochloride, levamisole hydrochloride, diphenhydramine hydrochloride, quinine, acetaminophen, ascorbic acid, caffeine, lactose, inositol, mannitol, glucose, sodium bicarbonate and calcium carbonate. In addition to the common, information-rich cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were used as these pulsed electroanalytical methods are typically considered more sensitive than CV and often employed for quantitative analyses of species present at low concentrations (Chapter 3). Overall, DPV resulted in voltammograms with peaks shaped closer to the ideal redox peaks, also referred to as ‘better defined’, thus enhancing the analytical performance of the assay. For example, In the analysis of diphenhydramine hydrochloride, DPV permitted the measurement of an oxidation with a peak displayed at 1.0 V vs Ag/AgCl, which was not observable when performing CV or SWV. On the other hand, SWV provided noticeably greater intensities of peak current, which allowed for a better detection of the difficult-to-observe redox reactions of quinine occurring at -0.4, 0.0 and 0.4 V vs Ag/AgCl. Some chemical species when present in seized drugs can alter the pH of the tested samples, such as ascorbic acid. Changes in pH will impact the redox activity of the pH-dependent electroactive species present in a sample, thus we investigated how pH of the solvent affected the observation of the redox peaks of those susceptible cutting agents, namely ascorbic acid, quinine, diphenhydramine hydrochloride, and levamisole hydrochloride (Chapter 4). Of particular interest was a significant change in the electrochemical characterization of these species when the pH was varied around their pKa values. Additionally, the composition of the solvent, or supporting electrolyte (SE) solution, can in some cases result in interactions with the analytes in the sample; the study of caffeine with different SE in Chapter 4 illustrates this situation. Specifically, sulfuric acid was the most suited SE of those tested for caffeine analysis. The impact of successive voltametric scans, on the analysis of chemical species were also investigated, using acetaminophen and quinine, demonstrating the development of additional redox peaks in some situations that could provide additional elements towards a more individualized electrochemical profile for cutting agents (Chapter 4). The influence of the material of the working electrode on the electrochemical characterization of cutting agents was explored. Solutions of ascorbic acid, acetaminophen, quinine, and diphenhydramine hydrochloride were electrochemically characterized using a glassy carbon and a platinum working electrode, while ascorbic acid was also characterized on gold and silver electrodes. These examples demonstrate the adaptability of this electroanalytical method with various commonly used electrodes. (Chapter 4). In Chapter 5, we applied similar electrochemical method to the identification of cutting agents and illicit drugs in two-component mixtures. Specifically, these trials included mixtures of fentanyl with a cutting agent at a relative ratio of 1 : 100, using as cutting agents ascorbic acid, diphenhydramine hydrochloride, or glucose, demonstrating the ability of this simple electrochemical method using common commercial electrodes to simultaneously detect illicit drugs and cutting agents.
Utilization of Blow Flies (Phormia regina) as Vertebrate Resource Diversity Indicators
(2022-08) Jones, Ashton Brooke; Picard, Christine; Walsh, Susan; Wells, Jeffrey
Blow flies are often utilized in the field of forensic science due to their ability to aid in the estimation of time since death. Currently, estimations of postmortem interval require assumptions to be made and are prone to a margin of error, prompting research that may contribute to more accurate postmortem interval estimations and help to fill in the gaps of unknown information. Blow flies are necrophagous, feeding on feces and carrion, and therefore, are constantly sampling the environment. This behavior can be exploited in order to monitor the biodiversity in an environment. Through analysis of DNA isolated from the guts of blow flies, information can be obtained regarding what animals have died in an environment, what animals are still living in that environment, and the abundance and diversity of the animals present in a specific environment. Using fly-derived ingested DNA is a viable method for vertebrate resource identification and biodiversity monitoring. Over the course of a two-summer sampling period, in and around two national parks, a total of 162 blow fly (Phormia regina) samples returned a positive vertebrate DNA identification, with 33 species identified from five animal orders. Of the total number of flies collected and analyzed, 23.58% returned a positive vertebrate species identification. The method detected both abundant and common species based on National Park surveys, as well as some uncommon or unknown to the park species. In the SE region, 9 individuals belonging to the Rodentia order, 12 individuals belonging to the Artiodactyla order, 21 individuals belonging to the Carnivora order, 1 individual belonging to the Cingulata order, and 3 individuals belonging to the Lagomorph order were detected. In the SE region, 63% of the individuals detected belonged to the common category, 14% of the individuals detected belonged to the uncommon category, and 23% of the individuals detected belonged to the not in park/unknown category. In the NW region, 42 individuals belonging to the Rodentia order, 46 individuals belonging to the Artiodactyla order, and 28 individuals belonging to the Carnivora order were detected. In the NW region, 52% of the individuals detected belonged to the abundant category, 36% of the individuals detected belonged to the common category, and 12% of the individuals detected belonged to the uncommon category. The relative biodiversity of the sampled environment can be inferred. In the SE region, the Shannon Biodiversity Index was calculated to be 2.28 with an evenness of 0.844, while in the NW region, the Shannon Biodiversity Index was calculated to be 2.79 with an evenness of 0.855. Unsurprisingly, there was greater biodiversity in the Northwest Park samples than in the Southeast Park samples. Additionally, the ideal weather conditions for blow fly collection were determined be at a temperature of between 60- and 80-degrees Fahrenheit, a relative humidity between 50% and 60%, no precipitation, and a wind speed between 2 and 8 miles per hour. This information has further implications in the field of forensic science, specifically dealing with wildlife forensics, pathogen distributions, and can help to improve accuracy in regards to postmortem interval (PMI) estimations.
The Detection and Identification of Explosives by Canines and Chemical Instrumentation
(2022-05) Reavis, Madison Dylan; Goodpaster, John V.; Manicke, Nicholas; Bors, Dana
With bombings in the United States on the rise for the first time since 2016, the detection and identification of explosives remains of pertinent interest to law enforcement agencies. This work presents two soon-to-be published research articles that focus on the detection and identification of explosives by both chemical instrumentation and canines. The first article, Quantitative Analysis of Smokeless Powder Particles in Post-Blast Debris via Gas Chromatography/Vacuum Ultraviolet Spectroscopy (GC/VUV), utilizes gas chromatography/vacuum ultraviolet spectroscopy (GC/VUV) to determine the difference in chemical composition of two smokeless powders in both pre- and post-blast conditions. The compounds of interest in this study were nitroglycerin, 2,4- dinitrotoluene, diphenylamine, ethyl centralite, and di-n-butyl phthalate. Concentration changes between pre- and post-blast smokeless powder particles were determined as well as microscopic differences between pre- and post-blast debris for both smokeless powders in all devices. To our knowledge, this is the first use of GC/VUV for the quantification of explosives. The second article, An Odor-Permeable Membrane Device for the Storage of Canine Training Aids, proposes the use of an odor-permeable membrane device (OPMD) as a standardized storage method for canine training aids. It is hypothesized that the OPMD would minimize cross-contamination between training aids, and that the OPMD could be used for canine training as well as storage. The goal of this research is to use flux and evaporation rate to quantify the explosive odor that escapes from the OPMD compared to unconfined explosives. Preliminary data suggests that there is an exponential relationship between relative boiling point and evaporation rate. It has been determined that compounds with higher boiling points have lower evaporation rates than compounds that have lower boiling points. The materials studied thus far are known odor compounds produced by explosive formulations. These include nitromethane, nitroethane, 1- nitropropane, r-limonene, and toluene.
Electrochemical Characterization of Fentanyl for Forensic Analysis
(2021-08) Sellnau, Natalie; Deiss, Frederique; Goodpaster, John; Manicke, Nicholas
The use and abuse of fentanyl has risen drastically over the last several decades. The abuse of this substance has created a hazardous situation for law enforcement and first responders because they could arrive at locations and not necessarily know that they will encounter fentanyl or a fentanyl analog. Fentanyl analogs are substances that have a similar structure to fentanyl, and while the analogs may have additional or altered groups on the molecule, the backbone structure remains similar. This work focus on the electrochemical characterization of fentanyl as a stepping stone for the detection of both fentanyl and later fentanyl analogs by electrochemistry. The metabolic reaction of fentanyl is an N-dealkylation to norfentanyl, occurring in the liver, and can be mimicked by electrochemistry through the irreversible oxidation of fentanyl. This electrochemical reaction is hypothesized to generate electroactive metabolites in solution. The combination of the visualization of both the irreversible oxidation with the development of the additional metabolic electrochemical peaks would constitute a unique electrochemical signature for fentanyl and fentanyl analogs towards a universal rapid screening assay. The electrochemical behavior of fentanyl was characterized in depth using multiple electrochemical techniques such as cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). The optimization of the supporting electrolyte, the potential range, and methods to decrease the background current were explored with CV. To work towards a more portable system, screen printed electrodes were used. The observation of the metabolic peaks remained challenging, and different methods were attempted to achieve it. The quantification of fentanyl was successfully demonstrated using the different electrochemical systems proposed in this work. The electrochemical characterization of fentanyl and the optimization of multiple experimental parameters were the first step in developing a universal, rapid, electrochemical sensing method for the detection of fentanyl and fentanyl analogs.

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