Evaluating Bioinformatic Pipeline Performance for Forensic Microbiome Analysis*,†,‡

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open-source bioinformatic pipelines, MG-RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha-diversity, and beta-diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low-abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications.     

DNA Recovery From Tape-Lifting Kits: Methodology and Practice

In the case of suspicious deaths, the technique of 1:1 taping is often used in Belgium. It consists of affixing a large number of adhesive tapes to the body of the victim. It is conventionally aimed at obtaining microtraces (e.g., fibers, hair) and is usually not used for DNA analysis. However, in some cases, DNA analysis of certain areas of interest identified on the 1:1 taping material can offer a last resort solution. The four-step method that is described in this article involves the selection of areas of interest on the body (Step 1), the selection of the corresponding tapes (Step 2), the decontamination of the tapes (Step 3), the selection of areas of interest on the tapes, for DNA sampling (Step 4). The method is illustrated by its successful application in four murder cases. In each case, DNA profiles of good quality could be identified, including profiles of persons different from the victim.     

Effect of Wind on Horizontal Displacement of Fatal Fall from a Height

The horizontal displacement of the human body resulting from fatal fall from a height is an important variable commonly used to inversely determine the cause or identify other forensic aspects of the fall. When examining the horizontal displacement, the wind effect is generally ignored. This technical note reports analytical modeling of the falling process, utilizing previous measurements of wind force acting on the human body, for determining the functional relationship between the wind speed and the horizontal displacement in the falling process. The result reveals that it does not take extremely rare wind conditions to cause a considerable shift of the human body, highlighting the importance to consider wind as a factor in investigations of fatal falls.     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.     

Volatile Organic Compound Profiling from Postmortem Microbes using Gas Chromatography–Mass Spectrometry

Volatile organic compounds (VOCs) are by-products of cadaveric decomposition and are responsible for the odor associated with decomposing remains. The direct link between VOC production and individual postmortem microbes has not been well characterized experimentally. The purpose of this study was to profile VOCs released from three postmortem bacterial isolates (Bacillus subtilis, Ignatzschineria indica, I. ureiclastica) using solid-phase microextraction arrow (SPME Arrow) and gas chromatography–mass spectrometry (GC-MS). Species were inoculated in headspace vials on Standard Nutrient Agar and monitored over 5 days at 24°C. Each species exhibited a different VOC profile that included common decomposition VOCs. VOCs exhibited upward or downward temporal trends over time. Ignatzschineria indica produced a large amount of dimethyldisulfide. Other compounds of interest included alcohols, aldehydes, aromatics, and ketones. This provides foundational data to link decomposition odor with specific postmortem microbes to improve understanding of underlying mechanisms for decomposition VOC production.     

Improved Area of Origin Estimation for Bloodstain Pattern Analysis Using 3D Scanning

An important component of crime scene reconstruction is bloodstain pattern analysis (BPA). Where BPA concerns impact patterns, estimating the area of origin is critical information for scene reconstruction. Traditionally, this is achieved by measuring individual bloodstains and performing trigonometric calculations; however, 3D scanning has been proposed as a viable alternative for overcoming logistical and practical concerns with the manual method. Therefore, this project aimed to establish whether the FARO Focus 3D scanner and FARO Zone 3D software can improve the accuracy of area of origin estimates relative to the manual method. We created a series of eight bloodstain impact patterns and performed paired analysis using the two methods to estimate areas of origin for each pattern. Our data suggested that FARO-derived estimates were generally more accurate than using the manual method. FARO-estimated heights of origin areas were generally closer to the true distance. Both methods underestimated the distance from the wall for most patterns originating 150mm or greater from the wall, but overestimated distances for patterns originating closer to the wall. The degree to which distances were underestimated increased significantly the further the blood source was from the wall and was greater for FARO-derived estimates. The results of this research contribute to the validation of these instruments for operational implementation for BPA and should be considered alongside the practical benefits of 3D scanning relative to manual methods. Further, 3D scanning can provide reliable BPA reconstruction documentation for technical review and court presentation.     

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex^® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex^® Fusion system at 29 cycles in a 10 μL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.     

A Comparison of Coronary Artery Stenosis Estimates Made by Forensic Pathologists and Medical Students

Previous studies suggest cardiovascular pathologists are less accurate than noncardiovascular pathologists (e.g., clinical pathologists) in estimating the degree of coronary artery stenosis. To further investigate the effect of training on accurate estimation of coronary artery stenosis, we designed a study to compare the accuracy of estimates made by forensic pathologists versus medical students. Six forensic pathologists and twelve medical students each independently examined 24 images of coronary artery cross sections and gave an estimate of the degree of stenosis. When comparing all 24 images, the forensic pathologists had a median difference between the estimated percentage of stenosis and actual percentage of stenosis of −12.380 and the medical students had a median difference of −16.50 (p-value of 0.08542). In estimating the percentage of stenosis, training in forensic pathology does not guarantee significantly improved accuracy compared with medical students. Our study showed no consistent statistically significant difference between estimates given by forensic pathologists and by medical students.     

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a “complete” Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.