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.

MaSTR™: an effective probabilistic genotyping tool for interpretation of STR mixtures associated with differentially degraded DNA.

The recently developed probabilistic genotyping software package MaSTR™ (SoftGenetics LLC) was used to develop statistical weight estimates for a variety of two-person STR mixture profiles with differentially degraded sources of DNA. A total of 864 analyses, on 144 two-person profiles, were performed. Mixture ratios ranged from 1:1 to 1:10, including pristine sources of DNA and various combinations of artificially degraded DNA (average size fragments of 150 or 250 bps). Quantities of DNA template were varied (0.1 to 0.5 ngs of total input) and MaSTR™ analysis was performed with eight chains of 10,000 or 40,000 iterations, with or without a conditioning profile to generate likelihood ratio (LR) values. Overall, the software performed as expected. The resulting log(LR) values for pristine mixture profiles were typically greater than 1030. Lower-quality mixture data associated with sources of DNA at ~ 0.05 ngs for each contributor resulted in peak imbalance and allelic dropout which reduced the weight in support of a contributor. This was exacerbated by higher levels of degradation, with some instances resulting in log(LR) values in support of an exclusion. These studies provide additional support for the use of probabilistic genotyping software solutions in forensic investigations, addressing concerns raised by the President's Council of Advisors on Science and Technology (PCAST).

High-throughput DNA sequencing of environmentally insulted latent fingerprints after visualization with nanoscale columnar-thin-film technique.

The goals of this study were to (a) ascertain human identity capabilities of DNA obtained from latent fingerprints that have been first environmentally insulted and then developed by the deposition of a columnar thin film (CTF), and (b) to determine if the CTF process and material are detrimental to single nucleotide polymorphism (SNP) analysis. Fingerprints were deposited on five different types of substrates and aged for one day, 7 days or 30 days while being environmentally insulted under one of the four conditions: 16.6 °C and 60% relative humidity (RH) (Condition A), 24.5 °C and 60% RH (Condition B), 35 °C and 67% RH (Condition C) and a cold condition (Condition D). Then CTF technique was then on 59% of these fingerprints. DNA samples from 805 fingerprints were extracted, quantified, subjected to manual library preparation using the Precision ID Identity Panel, and underwent high-throughput sequencing. The Ion S5™ platform was employed to sequence 124 SNP amplicons. SNPs were successfully sequenced from 802/805 samples. Total read depth was consistent across environmental conditions, and majority of samples had 100% profile completeness and 100% concordance. Anecdotally, libraries that were amplified with a higher cycle number had more 'Major Allele Frequency' flags compared to samples amplified with 23 cycle numbers, possibly due to stochastic effects. Neither the substrates nor the CTF process and materials inhibit downstream DNA analysis. DNA of low quality and quantity from the chosen samples can be sequenced using the Precision ID Identity Panel on the Ion S5™ platform which performed well, however, a different approach may be needed if spurious alleles are suspected.

Massively parallel sequencing and STR analysis from partial bloody fingerprints enhanced with columnar thin films.

Fingerprint enhancement often includes either physical or chemical approaches, such as fingerprint powder or cyanoacrylate fuming, to improve the quality of a fingerprint for visualization and analysis. However, these methods become more complex when fingerprints are partial bloody, and these procedures may interfere with downstream DNA analysis. Columnar thin film (CTF) deposition is a type of nanotechnology that utilizes an evaporant material to enhance a fingerprint under low-pressure conditions. Short tandem repeat (STR) analysis is the traditional method employed in crime laboratories. When DNA is of poor quality and quantity, like that often obtained from fingerprints, little to no genetic information may be obtained. Single nucleotide polymorphisms (SNPs) may be used to glean additional information when STR analysis fails. In this pilot study, 100 partial bloody fingerprints were collected from two donors and deposited on five different crime scene substrates, in which half were enhanced with CTFs and were graded for quality by an IAI-certified latent fingerprint examiner. CTF-developed fingerprints, on average, had higher grades compared to non-developed partial bloody fingerprints. STR analysis using Fusion 6C was performed to assess inhibition from the evaporant materials, in which no inhibition was observed. Sequencing of SNPs using the Precision ID Identity Panel was also employed, in which genetic information that could not be obtained from STRs was acquired with SNPs. Various sample types (i.e. pristine, low quality, and contaminated) utilized in this project demonstrated the acceptable performance of the Precision ID Identity Panel.

Determination of human identity from Anopheles stephensi mosquito blood meals using direct amplification and massively parallel sequencing.

DNA obtained from biological evidence can link individuals to a crime scene. DNA is typically obtained from body fluids deposited on various substrates such as fabric or common household objects. However, other unusual sources of human biological material can also be used to generate DNA profiles. Here we show that short tandem repeat (STR) DNA profiles can also be obtained from single source and mixtures of human DNA in the blood meals of Anopheles stephensi mosquitoes. Using direct amplification with the PowerPlex® Fusion 6C System, we have determined that full and partial profiles can be obtained by assessing degradation of DNA at various times post-feed up to 20-24 h post-blood meal. Moreover, we can assign donor identity through both STR profiles, as well as through single nucleotide polymorphisms (SNPs) detected using massively parallel sequencing (MPS) with the Precision ID Identity Panel and Ion Chef™/Ion S5™ System up to 24-48 h post-blood meal. Based on the results from a total of 490 mosquitoes fed on 11 different sources of human blood, we conclude that both STR and SNP technologies can be applied to mosquito blood meals as effective forensic approaches to determine the identity of specific individuals and establish the timing of their presence at a crime scene.