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Forensic Science International. Genetics Jul 2021In the past decade, hybridization capture has gained attention within the forensic field for its possible use in human identification. One of the primary benefits to... (Review)
Review
In the past decade, hybridization capture has gained attention within the forensic field for its possible use in human identification. One of the primary benefits to capture enrichment is its applicability to degraded DNA fragments that, due to their reduced size, are not amenable to traditional PCR enrichment techniques. Hybridization capture is typically introduced after genomic library preparation of extracted DNA templates for the subsequent enrichment of mitochondrial DNA or single nucleotide polymorphisms within the nuclear genome. The enriched molecules are then subjected to massively parallel sequencing (MPS) for sensitive and high-throughput DNA sequence generation. Bioinformatic analysis of capture product removes PCR duplicates that were introduced during the laboratory workflow in order to characterize the original DNA template molecules. In the case of aged and degraded skeletal remains, the fraction of endogenous human DNA may be very low; therefore low-coverage sequence analysis may be required. This review contains an overview of current capture methodologies and the primary literature on hybridization capture as evaluated for forensic applications.
Topics: DNA Fingerprinting; DNA, Mitochondrial; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Nucleic Acid Hybridization; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 33770700
DOI: 10.1016/j.fsigen.2021.102496 -
Forensic Science International. Genetics Mar 2023Several studies have demonstrated that DNA can be indirectly transferred from an individual onto a surface. Therefore, the presence of DNA that is compatible with a...
Several studies have demonstrated that DNA can be indirectly transferred from an individual onto a surface. Therefore, the presence of DNA that is compatible with a given person does not necessarily mean that this person has touched the surface on which the DNA was recovered. The present work simulates cases, where DNA is recovered on a door handle and compared to several reference DNA profiles. The DNA profile of the trace shares DNA components with a person of interest (POI). When asked about the DNA results, the POI says he has nothing to do with the incident and has never been at the scene. However, a possibility would be that the DNA came from his recently stolen gloves. Someone else, the alternative offender (AO), could have opened the door wearing his gloves (POI's gloves), and transferred his DNA (POI's DNA). Based on the above-mentioned scenario, 60 burglary simulations experiments were carried out to generate data to assess DNA results given these allegations. The quantity and quality of DNA profiles (NGM SElect) recovered when the POI opened/closed the door bare-handed or when someone else performed the same activity but using POI's gloves, were compared. The gloves were regularly worn during at least three months by their owner during the winter. On the contrary, the AO wore them only for two minutes. Among the traces collected on the door handles, less than 50% of the traces led to interpretable DNA profiles. In 30% of the cases (3/10), when the door was opened/closed with bare hands, the DNA found on the door handle led to a mixed DNA profile with the POI's DNA aligning with the major contributor. For the experiments where the AO opened/closed the door with the POI's gloves, the POI's DNA was compatible with 22% (11/50) of the mixed DNA profile, aligning with the major in 8% of the cases (4/50). The DNA profiles of the offices' occupants were observed on the door handles, but not the AO's. In addition to the results of the experiments, we show two examples of how one can assess results observed in casework. Given the possibility of indirect transfer of minute DNA quantities, this research emphasizes the need to evaluate DNA results given the activities when the POI has a legitimate reason that can explain the presence of their DNA.
Topics: Humans; Male; DNA Fingerprinting; Touch; DNA; Hand; Criminals
PubMed: 36563530
DOI: 10.1016/j.fsigen.2022.102823 -
Forensic Science International. Genetics Sep 2022In sexual assault cases, it can be challenging to identify the type of body fluids/ cell types present in a crime scene sample, especially the origin of epithelial...
In sexual assault cases, it can be challenging to identify the type of body fluids/ cell types present in a crime scene sample, especially the origin of epithelial cells. Therefore, more labs are applying mRNA body fluid analysis for saliva, skin and vaginal mucosa markers. To address activity level propositions, it is necessary to assign probabilities of transfer, persistence, prevalence and recovery of DNA and mRNA markers. In this study we analysed 158 samples (fingernail swabs, penile swabs and boxershorts) from 12 couples collected at different time points post intimate contact and after non-intimate contact in order to detect DNA from the person of interest (POI) and mRNA vaginal mucosa markers. Samples were DNA and RNA co-extracted and analysed with PowerPlex®Fusion 6C System and 19-plex mRNA primer mix respectively, using Endpoint PCR and the CE platform. Vaginal mucosa was detected up to 36 h post intimate contact, but also detected in one non-intimate contact sample. In 94% of intimate contact and 50 % of non-intimate contact samples the DNA results support the proposition that POI is the donor (LR ≥ 10,000). There was a strong association between the detection of vaginal mucosa and the average RFU value of the POI. The data were used to instantiate a comprehensive Bayesian network to evaluate the evidence at activity level, given alternate propositions conditioned upon indirect or direct transfer events. It is shown that the value of the evidence is mainly affected by the high DNA quantity (measured as mean RFU) that is recovered from the POI. The detection of vaginal mucosa had low impact upon the resultant likelihood ratio.
Topics: Bayes Theorem; DNA; DNA Fingerprinting; Female; Humans; Mucous Membrane; RNA, Messenger
PubMed: 35914368
DOI: 10.1016/j.fsigen.2022.102750 -
Legal Medicine (Tokyo, Japan) Feb 2021The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing...
The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing success is often hampered by low amounts and strong degradation of nuclear DNA. Common strategies consist of quantitative polymerase chain reaction (qPCR)-based analysis of the abundance of a short versus a long nuclear amplicon, the latter prone to DNA degradation. To increase sensitivity, commercial qPCR solutions rest on amplification of multi-copy DNA sequences. Here we show that ribosomal DNA (rDNA) sequences are well suited for the same purpose. Because rDNA sequences are present in high copy number in most eukaryotic species, qPCR strategies can easily be adapted to non-human species. In this paper, we establish qPCR-based assays for human or dog DNA, respectively, which allow for sensitive analysis of DNA amounts and DNA degradation. We show that the human system can be applied to DNA of single telogen hairs, where STR typing success correlates with measured amounts and integrity of the DNA. By adapting the system to dog rDNA sequences we found that single telogen dog hairs often displayed less DNA degradation than human telogen hairs, in most cases allowing for successful STR typing. Thus, qPCR-based analysis of rDNA represents a cost-effective, highly sensitive strategy to assess DNA amount and integrity that can be adapted to hairs or other traces from various animal species.
Topics: Animals; DNA Fingerprinting; DNA, Ribosomal; Dogs; Forensic Genetics; Hair; Humans; Microsatellite Repeats; Real-Time Polymerase Chain Reaction
PubMed: 33248354
DOI: 10.1016/j.legalmed.2020.101819 -
Scientific Reports Jul 2021We address computational and statistical aspects of DNA-based identification of victims in the aftermath of disasters. Current methods and software for such...
We address computational and statistical aspects of DNA-based identification of victims in the aftermath of disasters. Current methods and software for such identification typically consider each victim individually, leading to suboptimal power of identification and potential inconsistencies in the statistical summary of the evidence. We resolve these problems by performing joint identification of all victims, using the complete genetic data set. Individual identification probabilities, conditional on all available information, are derived from the joint solution in the form of posterior pairing probabilities. A closed formula is obtained for the a priori number of possible joint solutions to a given DVI problem. This number increases quickly with the number of victims and missing persons, posing computational challenges for brute force approaches. We address this complexity with a preparatory sequential step aiming to reduce the search space. The examples show that realistic cases are handled efficiently. User-friendly implementations of all methods are provided in the R package dvir, freely available on all platforms.
Topics: DNA Fingerprinting; Disaster Victims; Female; Humans; Male; Pedigree; Probability; Software
PubMed: 34211052
DOI: 10.1038/s41598-021-93071-5 -
Journal of Visualized Experiments : JoVE Jul 2021In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and...
In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences. This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard. Following this guide, students should be able to: • Harvest and extract DNA from buccal mucosa epithelial cells • Perform a PCR experiment and understand the function of various reaction components • Analyze the amplicons by agarose gel electrophoresis and interpret the results • Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences.
Topics: Alleles; DNA Fingerprinting; Humans; Laboratories; Minisatellite Repeats; Paternity; Phylogeny
PubMed: 34338668
DOI: 10.3791/62305 -
Forensic Science, Medicine, and... Jun 2023We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI...
We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI (4',6-diamidino-2-phenylindole). Knowledge of the presence and number of osteocytes is key to any success in subsequent DNA profiling. Osteocytes are most numerous cells and thus the main source of DNA in bone samples, which can be preserved for histological analyses. Archived samples are either fixed in formalin or preserved in ethanol prior to embedding in resin. These resin-embedded samples are potentially used as ante mortem reference samples. Cases of a missing person investigation are one example where this type of preserved reference material may be of value. When resin is required for sample preservation it represents a problem for subsequent DNA profiling, if needed as a reference sample in human identification. It is essential therefore to remove the resin prior to DNA analyses as resin is a known inhibitor of DNA profiling. Current methods of resin removal are lengthy and require toxic chemicals. This report describes a simplified process to remove resin and visualise the location of nucleated osteocytes. Eight sections of bone samples at 5-µm thickness were stained with DD and DAPI. A further three samples were processed using a formalin-fixed method and three additional samples treated following an ethanol-preserved method (11 samples for both the formalin-fixed and 11 for the ethanol-preserved with eight in common). The location and number of nuclei could be recorded clearly due to the fluorescence created by the dye binding to DNA. The number of stained nuclei correlated with the mass of DNA isolated from the sections (r = 0.873, p = 1.21 × 10). A significant difference between the degradation indices of two groups (p = 8.505 × 10) showed that ethanol preservation is a preferred method to yield DNA of the quality needed for subsequent short tandem repeats (STR) profiling. Ten of the 11 samples isolated using the ethanol-preserved process recorded a complete STR profile (30/30 alleles), whereas eight of the formalin-fixed samples generated full profiles, and only one of the 11 samples amplified less than 23 alleles. Both the ethanol-preserved and formalin-fixed methods are an improvement on current methods by removing the need for strong solutes in resin removal, and the method leads to STR profiles from resin-embedded bone samples within 24 h.
Topics: Humans; Osteocytes; DNA; Formaldehyde; Bone and Bones; Coloring Agents; Ethanol; DNA Fingerprinting; Microsatellite Repeats
PubMed: 36401783
DOI: 10.1007/s12024-022-00559-1 -
Journal of Forensic Sciences Jul 2022The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although...
The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor-intensive and inefficient regarding time and sperm DNA recovery - hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high-quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP-40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP-40 methods to efficiently lyse spermatozoa and provide high-quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high-quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.
Topics: DNA; DNA Fingerprinting; Humans; Male; Microsatellite Repeats; Semen; Sex Offenses; Specimen Handling; Spermatozoa
PubMed: 35285573
DOI: 10.1111/1556-4029.15027 -
Methods in Molecular Biology (Clifton,... 2021Recent scientific advancements in the field of genetics have fostered significant changes for the criminal justice system. Growing National DNA databases, public DNA...
Recent scientific advancements in the field of genetics have fostered significant changes for the criminal justice system. Growing National DNA databases, public DNA databases, private direct-to-consumer (DTC) DNA testing companies, and improvements in next-generation sequencing (NGS) have resulted in effective methods for tracking down criminals and exonerating the innocent. While these recently discovered and profound techniques seem to provide benefits, their use in forensic detection has become subject to harsh legal opposition. Ultimately, should law enforcement be permitted to analyze DNA found at crime scenes and DNA that has accumulated in national, public, and private databases to aid in their investigations, or are individuals' privacy rights breached in the process?
Topics: DNA; DNA Fingerprinting; Data Analysis; Data Collection; Data Management; Databases, Nucleic Acid; Forensic Genetics; Genetic Techniques; High-Throughput Nucleotide Sequencing; Humans
PubMed: 33606268
DOI: 10.1007/978-1-0716-1103-6_19 -
International Journal of Legal Medicine Jul 2023Recovery of suitable amounts of DNA from ammunition cartridges for short tandem repeat (STR) or mitochondrial (mt) DNA analysis has been a challenge for crime...
Recovery of suitable amounts of DNA from ammunition cartridges for short tandem repeat (STR) or mitochondrial (mt) DNA analysis has been a challenge for crime laboratories. The metal composition of cartridge cases and projectiles exposes the DNA to harmful ions that damage and ultimately degrade the DNA such that it cannot be effectively amplified. The current study assessed the impact of time and storage conditions on touch DNA deposited on cartridge components of varying metal content: aluminum, nickel, brass, and copper. Elevated humidity levels facilitated greater DNA degradation and loss compared to low humidity (or "dry") conditions, indicating that recovered cartridge component evidence should be stored in a low-humidity environment immediately after collection, preferably with a desiccant. As expected, a relationship was observed between the amount of time elapsed since the cartridge components were handled and the associated DNA yield. Interestingly, while yields dropped considerably in the first 48-96 h post-handling, regardless of the storage conditions, a layering effect was observed that helps maintain a relatively constant level of surface DNA over extended periods of time. An apparent layering effect was also observed on cartridge components following multiple surface depositions, where yields were two times higher than single deposition samples at similar timepoints. Overall, these findings suggest that storage conditions and a layering affect play an important role in the preservation of DNA on ammunition components.
Topics: Humans; DNA Fingerprinting; DNA, Mitochondrial; Touch; Specimen Handling
PubMed: 37237149
DOI: 10.1007/s00414-023-03018-w