Forensic DNA Analysis Research Articles

Unlocking Secrets: Bioinformatics' Impact on Forensic Bio-Examinations

Bioinformatics (BI) is a diverse and interdisciplinary domain that studies biological data through computational techniques. Its rapid technological progress has led to a significant increase in biological data production, sparking a notable expansion in BI research and application. In recent years, the convergence between BI and forensic science has become increasingly evident, with the growing use of BI tools and techniques in forensic investigations. BI is not only extensively used in biomedical research to analyse biological data and forecast gene regulation but also in the development of drugs targeting disorders linked to epigenetic alterations. It merges principles from various fields like biology, computer science, and statistics to process extensive omics data, create databases, predict structures, and model metabolic processes. The impact of BI on biomedical research is substantial, aiding in data processing, prediction, and visualisation. Moreover, BI is increasingly important in analysing data generated through Sanger-based electrophoresis methods and massively parallel sequencing in forensic DNA analysis, demonstrating its practical application in the field. Therefore, BI plays a critical role in forensic identification, searching databases side by side, forensic DNA phenotyping and biographic ancestry, forensic genetic genealogy, and forensic epigenomics. An overview of different bioinformatic methodologies employed in forensic applications, exploring approaches for managing data and investigating trend analysis strategies within forensic contexts, is discussed. Additionally, it investigates data management strategies. The primary objective was to explore how BI facilitates forensic BI's use, integration, and impact. Utilising a systematic literature review and content analysis, the study examined peer-reviewed articles, conference proceedings, and reports from critical databases like Google Scholar and Scopus, focusing on publications from 2010 to 2024. Key findings highlight the significant advancements in genomic sequencing technologies and their application in personalised therapeutics. Forensic scientists can improve forensic exams' accuracy, reliability, and efficiency by using BI tools, metrics, and efficient data management procedures.

Evaluation of the usefulness of insertion-null markers in critical skeletal remains

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

Precision touch DNA sampling on plastic bag knots for improved profiling of packer and holder contributions

In forensic DNA analysis, evidence sampling stands as a pivotal step setting the ground for the quality of the forensic profiling. The collection of touch DNA from objects, when guidelines are scarce or absent, is usually governed by ad hoc decisions based on the available case circumstances. In our laboratory, in the context of illicit drug-related crimes, similar objects are frequently encountered, offering an opportunity for the standardization of evidence treatment. This study aims to develop an effective method for sampling touch DNA from knots on plastic bags. We examine both the exposed and hidden areas of knots, considering the latter as "protected" zones less likely to accumulate biological material during subsequent handling. The study contrasts a single sample method (whole knot surface sampling, Method 1) with dual-sample methods that separate exterior (exposed) and interior (hidden) surfaces of the knot. Notably, our study consistently reveals higher DNA yields from exterior surfaces of the knots as opposed to interior samples. Importantly, our findings demonstrate that utilizing a single sample may produce DNA profiles that are not interpretable, while employing a dual-sample approach may allow for the differentiation between the genetic contributions of the person who tied the knot, the packer, from the person who held the package, the holder. We have refined the dual-sample method to reduce holder DNA in the interior sample while maintaining it on the exterior, also allowing the packer's DNA to be detected on both surfaces. We explore four dual-sample collection methods. Method 2 involves taking the first sample from the exterior and the second from the interior of an untied knot. Method 3 visually differentiates between the original exposed and hidden surfaces for precise sampling. Method 4 employs tools to open the knot for interior sampling. Method 5 uses Diamond dye to highlight cell-free DNA on both surfaces before sampling. In conclusion, this study not only clarifies the complex dynamics of touch DNA transfer and collection on plastic bag knots, but also offers insights into standardizing evidence collection in similar cases.

Forensic DNA Profiling: Legal and Ethical Considerations

Forensic DNA analysis is vital in criminal investigations, posing intricate legal and ethical dilemmas. This emphasises the need to balance DNA technology benefits with individual rights, prioritizing privacy, consent, and ethical practices. Stringent regulation of DNA data management is essential to prevent misuse and ensure privacy protection. Advances like probabilistic genotyping software improve interpretation accuracy, addressing complexities in mixture analysis. Responsible DNA use also requires navigating emerging technologies like next-generation sequencing while upholding ethical standards. Establishing transparency, accuracy, and ethical conduct throughout the analysis process is crucial for building public trust. Regulatory oversight and accountability mechanisms are necessary to address errors and maintain fairness in DNA data management. Through continuous dialogue and adherence to ethical standards, the forensic community can uphold scientific integrity and preserve public confidence in DNA analysis.

Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control.

The negative template control or negative amplification control has been an essential component of the forensic DNA analysis workflow that helps monitor contamination. As such, the inclusion of a negative control in forensic DNA analysis has been a requirement for all laboratories audited under the FBI's Quality Assurance Standards. As massively parallel sequencing (MPS) becomes more conventional in forensic laboratories, considerations for the inclusion of a negative control in every sequencing run can be evaluated. Although the inclusion of a negative control in library preparation and the first sequencing run has a practical function, there is less utility for its inclusion in all subsequent sequencing runs for that library preparation. Although this is universal to all MPS assays, it is most relevant for an assay that has a low sample multiplexing capacity, such as the ForenSeq Kintelligence Kit (Qiagen/Verogen, Inc.). The ForenSeq Kintelligence Kit is an investigative genetic genealogy (IGG) sequencing-based assay that targets 10,230 forensically relevant single-nucleotide polymorphisms. The manufacturer recommends multiplexing 3 libraries per sequencing run, which includes controls. The purpose of this study was to investigate the effect of the inclusion of a negative control in every Kintelligence sequencing run. We observed that the library generated from a negative amplification control will take 7%-14% of the run output. The loss of sequencing space taken by a negative control decreased the available output for DNA-containing samples, leading in some cases to allele or locus dropout and accompanying higher numbers of sixth to seventh order unknown associations in GEDmatch PRO.

Assessing the feasibility of free DNA for disaster victim identification and forensic applications

In tropical disaster victim identification (DVI) scenarios, challenging environmental conditions lead to accelerated DNA degradation in remains. To further enhance the utilization of leached DNA from tissue in the preservative solution (termed “free DNA”) as an alternative source, we incorporated new results by assessing its integrity in postmortem and decomposing cadavers preserved in DNA/RNA Shield™ and modified TENT, with silica-based purification (QIAquick®) for faster processing. The psoas muscle tissues of one decomposed and ten cadavers were preserved in each solution at 25 °C and 35 °C for 3 months. Free DNA efficiency was compared with individual reference samples for reliable results in quantity, quality, and STR profiles. The findings revealed that DNA/RNA Shield™ effectively preserves free DNA integrity for extended storage, while modified TENT is more suitable for short-term storage due to higher degradation levels. Moreover, the use of free DNA samples with massive parallel sequencing displays potential for forensic DNA analysis. Successful amplification of the mtDNA control region enables variant calling and heteroplasmy analysis while also serving as quality control using ACTB and enabling differentiation within the 16S rRNA region for microbiome analysis. The simplicity of handling free DNA for PCR-based forensic analysis adds to its potential for various applications, including DVI and field-based analysis of biological evidence.

Sequence analysis and secondary structure prediction of autosomal STR alleles using next generation sequencing (NGS) data

The inception of NGS technology in forensic DNA analysis explores the sequence-based alleles in STR markers. This allows the in-depth analysis of observed STR allelic sequences to understand their nature, stability, inheritance, and possible generation of artifacts during their analysis. In the current study, 100 allelic sequences at 20 STR markers generated using the NGS technique were analyzed for their sequence topographies and prediction of secondary structures. The G + C content of the alleles observed in 20 STR markers used in this study varied from 58.65 ± 1.367% (D2S1338) to 7.62 ± 0.844% (D12ATA63). The average exact mass of one stand and for the opposite stand mass was found to be highest in FGA (25,963.248 ± 1623.271; 27,501.720 ± 1712.691), whereas, D4S2408 generated the lowest exact mass of one stand and for the opposite stand mass (11,136.354 ± 1521.757; 11,582.021 ± 1585.486). As expected, none of the STR markers showed the presence of open reading frames, Codons, and CRISPR sequences. Three STR markers viz. D2S1338, TH01, and D5S2800 showed the presence of restriction sites for Cac8I, TspGWI, TspDTI, AccI, and Hpy8I enzymes. Phylogenetic analysis reveals the close association of alleles between the D12ATA63 and D19S433 markers. Stable pseudoknots were predicted at alleles of D2S1338 showing an average energy of −0.76 with the highest number of nucleotides present in the pseudoknots i.e., 21.33, suggesting this marker is more prone to generate amplification artifacts.

Targeted enrichment of whole-genome SNPs from highly burned skeletal remains.

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole-genome single-nucleotide polymorphism (SNP) quality and quantity using next-generation sequencing (NGS). We present whole-genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double-stranded DNA libraries then enriched for whole-genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed-effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole-genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification.

Homologous blood transfusion and doping: Where are we now?

Homologous blood transfusion (HBT) is used for doping in endurance sports since the 1960s. The blood comes from a compatible donor, that is, someone with a compatible ABO and rhesus blood group. Despite been prohibited by the IOC in 1985, no detection method was available until 2003. Then came the idea to use red blood cells (RBC) minor blood groups antigens that constitute an "identity" card of someone's RBC to detect the presence of a second RBC population. The method validated for doping control samples uses flow cytometry after incubation of isolated RBC with eight to 12 primary antibodies against specific minor blood groups antigens. The presence of double populations of RBC is revealed by a major and a minor peak in a fluorescence histogram. The sensitivity was estimated sufficient to detect HBT for a few weeks. Despite the complexity and cost of the method, right after its application in 2004, several cases of HBT were identified but the number of cases dropped rapidly over the years. In the 2010s, other ways to detect HBT were developed and evaluated: indirect detection using the Athlete Biological Passport approach, and a few years later forensic DNA analysis to establish the presence of two different DNA in a blood sample after HBT. Despite the high specificity of the latter, the sensitivity was recently questioned in vivo. Nowadays, the flow cytometry method remains the method of choice for HBT detection and recent investigations helped to simplify the method and increase its specificity and sensitivity.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1–6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

Identification of the efficacy of parentage testing based on bi-allelic autosomal single nucleotide polymorphism markers in Taiwanese population.

Parentage testing is crucial for forensic DNA analysis, using short tandem repeats (STRs). Single nucleotide polymorphisms (SNPs) with high minor allele frequency (MAF) are promising for human identification. This study aimed to develop SNP markers for parentage testing in the Taiwanese population and compare their accuracy with STRs. The TPMv1 SNP microarray (714,457 SNPs) was used to screen 180,000 Taiwanese individuals and analyze the SNP data using PLINK. After quality control, allelic distribution, and MAF considerations, a set of SNPs with significant inheritance information was selected. Parentage testing was conducted on 355 single parent-child pairs using both STRs and SNPs, employing three kinship algorithms: identity by descent, kinship-based inference for genome-wide association studies, and the combined paternity index/probability of paternity (CPI/PP). An Affymetrix signature probe for kinship testing (ASP) was also used. Based on the quality control and selection criteria, 176 SNPs with MAF > 0.4995 were selected from the Taiwanese population. The CPI/PP results calculated using SNPs were consistent with the STR results. The accuracy of the SNPs used in the single-parent-child parentage testing was > 99.99%. The set of 176 SNPs had a higher identification rate in the single parent-child parentage test than in the ASP. The CPI/PP value calculated using 176 SNPs was also more accurate than that calculated using ASP. Our findings suggest that these 176 SNPs could be used for single-parent-child parentage identification in the Taiwanese population.

Optimizing Analytical Thresholds for Low-Template DNA Analysis: Insights from Multi-Laboratory Negative Controls.

When analyzing challenging samples, such as low-template DNA, analysts aim to maximize information while minimizing noise, often by adjusting the analytical threshold (AT) for optimal results. A potential approach involves calculating the AT based on the baseline signal distribution in electrophoresis results. This study investigates the impact of reagent kits, testing quarters, environmental conditions, and amplification cycles on baseline signals using historical records and experimental data on low-template DNA. Variations in these aspects contribute to differences in baseline signal patterns. Analysts should remain vigilant regarding routine instrument maintenance and reagent replacement, as these may affect baseline signals. Prompt analysis of baseline status and tailored adjustments to ATs under specific laboratory conditions are advised. A comparative analysis of published methods for calculating the optimal AT from a negative signal distribution highlighted the efficiency of utilizing baseline signals to enhance forensic genetic analysis, with the exception of extremely low-template samples and high-amplification cycles. Moreover, a user-friendly program for real-time analysis was developed, enabling prompt adjustments to ATs based on negative control profiles. In conclusion, this study provides insights into baseline signals, aiming to enhance genetic analysis accuracy across diverse laboratories. Practical recommendations are offered for optimizing ATs in forensic DNA analysis.

Analysis of maternal genetic structure of mitochondrial DNA control region from Tai-Kadai-speaking Buyei population in southwestern China

BackgroundEven though the Buyei are a recognised ethnic group in southwestern China, there hasn’t been much work done on forensic population genetics, notably using mitochondrial DNA. The sequences and haplogroups of mitochondrial DNA control regions of the Buyei peoples were studied to provide support for the establishment of a reference database for forensic DNA analysis in East Asia.Methods and resultsThe mitochondrial DNA control region sequences of 200 Buyei individuals in Guizhou were investigated. The haplotype frequencies and haplogroup distribution of the Buyei nationality in Guizhou were calculated. At the same time, the paired Fst values of the study population and other populations around the world were computed, to explore their genetic polymorphism and population relationship. A total of 179 haplotypes were detected in the Buyei population, with frequencies of 0.005–0.015. All haplotypes were assigned to 89 different haplogroups. The haplotype diversity and random matching probability were 0.999283 and 0.0063, respectively. The paired Fst genetic distances and correlation p-values among the 54 populations revealed that the Guizhou Buyei was most closely related to the Henan Han and the Guizhou Miao, and closer to the Hazara population in Pakistan and the Chiang Mai population.ConclusionsThe study of mitochondrial DNA based on the maternal genetic structure of the Buyei nationality in Guizhou will benefit the establishment of an East Asian forensic DNA reference database and provide a reference for anthropological research in the future.

Emerging Technologies in Forensic DNA Analysis

Emerging Technologies in Forensic DNA Analysis

Emerging Technologies in Forensic DNA Analysis

Emerging Technologies in Forensic DNA Analysis

Y chromosome haplogroup N in a Japanese population is classified into three subclades, and two DYS385 loci, a duplicated Y-STR, are duplicated again in subclade N-M1819

DYS385 is one of the major Y chromosome short tandem repeats (Y-STRs) in forensic genetics and exists as 2 copies in the human Y chromosome palindrome P4 region. In this study, we found that some samples were estimated to have ≥ 4 copies of DYS385 in Y chromosome haplogroup N in a Japanese population. Y chromosome haplogroup N is distributed widely in eastern/central Asia, Siberia, and eastern/northern Europe, and is also observed in Japan; however, little is known about haplogroup N subclades in the Japanese population. To reveal the link between increased DYS385 copy number and haplogroup N subclades, we sequenced single nucleotide polymorphisms to classify the subclades. As a result, the Japanese Y chromosomes of haplogroup N were classified into three subclades, and an increased DYS385 copy number was specific to subclade N-M1819* (N1b2*). These results are of use in forensic DNA analysis because Y-STR copy number is important for the interpretation of Y-STR typing results of male DNA mixtures and kinship analysis. We also found that DYS458.1 microvariants (DYS458 intermediate alleles with single-nucleotide insertion) were observed only in subclade N-CTS962 (N1b1b∼) samples. Given that previous studies reported that DYS458.1 microvariants are observed in Y chromosomes of haplogroup N in other populations, DYS458.1 might be used to infer haplogroup N subclades without limitation to the Japanese population. The results of this study will be beneficial not only to forensic genetics but also to anthropological studies.

Comparative Study of Two Semi-automated Forensic DNA Extraction Methods

Automation in forensic DNA analysis is crucial for analysts to reduce time, improve results, and decrease risk of contamination. With the variety of commercially available automated DNA extraction systems, comes the need for end-users to be informed of what they provide and what they might lack. Thus, this study aimed to evaluate the efficiency of two semi-automated DNA extraction systems used for forensic DNA analysis: Automate Express™ and Hamilton Microlab STAR™ system, for four parameters; reproducibility, stability, sensitivity and contamination. Overall, the results indicated that both semi-automated systems performed similarly in providing robust and reproducible DNA results while maintaining good capability to overcome PCR inhibition with low risk of contamination. The two semi-automated systems showed higher DNA recovery than organic extraction using phenol-chloroform by 22% for semen and 7% for blood samples. In addition, three sample types, blood, saliva, semen were tested to compare the two systems (total samples n=100). Overall, the data showed the average DNA recovery for Hamilton was higher than the DNA recovery by Automate Express™ for the blood and semen sample types indicating better performance of the Hamilton Microlab STAR™ in terms of recovery and sensitivity level.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

Developmental validation of a custom-designed Multi-InDel panel: A five-dye multiplex amplification system for challenging DNA samples.

The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study, we have introduced a custom-designed panel capable of simultaneously amplifying 41 novel Multi-insertion/deletion (Multi-InDel) markers and an amelogenin locus using the capillary electrophoresis platform. Through a developmental validation study conducted in accordance with guidelines recommended by the Scientific Working Group on DNA Analysis Methods, we demonstrated that the new Multi-InDel system exhibited the sensitivity to produce reliable genotyping profiles with as little as 62.5 pgof template DNA. Accurate and complete genotyping profiles could be obtained even in the presence of specific concentrations of PCR inhibitors. Furthermore, the maximum amplicon size for this system was limited to under 220 bp in the genotyping profile, resulting in its superior efficiency compared to commercially available short tandem repeat kits for both naturally and artificially degraded samples. In the context of mixed DNA analysis, the Multi-InDel system was proved informative in the identification of two-person DNA mixture, even when the template DNA of the minor contributor was as low as 50 pg. In conclusion, a series of performance evaluation studieshave provided compelling evidence that the new Multi-InDel system holds promise as a valuable tool for forensic DNA analysis.

The Boundary of Artificial Intelligence in Forensic Science

Artificial intelligence (AI) is poised to revolutionize forensic science by enhancing evidence analysis and aiding in crime-solving. This paper explores AI applications in forensic fields, focusing on DNA mixture analysis, the cause of death diagnosis, and the challenges AI faces in forensic science. It addresses the limitations of AI in forensic science, emphasizing the necessity for transparent AI systems used ethically. Forensic errors are often not due to misclassification by forensic scientists but stem from incompetence, fraud, weak scientific foundations, or organizational deficiencies, resulting in wrongful convictions. Systemic issues like dependence on presumptive tests and inadequate defense contribute to errors. Inconsistencies plague various domains of forensic science, including fingerprint and DNA analysis. Establishing standardized analytical procedures can enhance consistency and credibility in forensic science. AI applications in forensic anthropology, pathology, genetics, and statistics are explored, with a focus on their potential and challenges.