Forensic Genetic Laboratories Research Articles

Cleaning protocols in forensic genetic laboratories.

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD4L did not.

Review of Six Different Next Generation Sequencing (NGS) Techniques for Forensic Science, including Advantages and Disadvantages

Next-generation sequencing methods have advanced greatly since Sanger sequencing, allowing for increased data yield, productivity, and utility. Read time can be used to categorize the upcoming technological generations. This article summarizes the differences between the two technological paradigms, the second-generation (short-read) kind, and the third-generation (long-read) variety. Popular technologies such as Ion Torrent and Illumina stand in for short-read sequencing methods, whereas Oxford Nanopore and Pacific Biosciences are used to represent long-read sequencing approaches. The introduction of the first next-generation sequencing (NGS) technology about ten years ago completely transformed the study of genetics. Whole genomes are now mapped and published practically weekly as a result of speed and cost advances. The number of scholarly papers and conference presentations highlighting the forensic uses of NGS in multiple forensic genetic laboratories has somewhat increased from the previous year. These results show that NGS provides new opportunities for forensic genomic investigation. To gather more information from multiple specimens in a single experiment, combinations of different markers, such as Short Tandem Repeats (STRs), Single Nucleotide Polymorphisms (SNPs), insertion/deletions, and mRNA, can be used instead of the usual Polymerase Chain Reactions- CE techniques. The most significant forensic STR loci's true spectrum of variation and hitherto unknown STR alleles have been discovered. We will address the possible use of singlemolecule sequencing and NGS in forensic science.

Application of the Investigator Argus X-12 QS Kit and the Famlinkx Software for Solving Complex Kinship Forensic Genetics Cases in Colombia: Case Report

X-chromosome short tandem repeats (X-STRs) genotyping is a powerful ¬tool in forensic genetics for resolving intricate kinship cases due to its distinctive inheritance pattern. It proves particularly valuable in scenarios where the application of autosomal (A-STR) and Y-chromosomal STR (Y-STR) markers falls short in addressing such cases, especially those involving complex situations and kinship analyses encompassing extensive and incomplete pedigrees. The recent advancement and implementation of the Argus X-12 QS kit along with the FamilinkX software for X-STR analysis have facilitated the resolution of forensic cases that were formerly inconclusive or not received by the laboratory due to their high complexity. This article delineates seven intricate cases of kinship and identification conducted at the Forensic Genetics Laboratory of the National Institute of Legal Medicine and Forensic Sciences in Bogotá, Colombia, in instances where autosomal STRs yielded non-conclusive or weak likelihood ratio (LR) values, the combination of the Argus X-12 QS kit and the FamilinkX software proved instrumental in elucidating and enhancing LR values, thereby leading to conclusive outcomes. The article details a case involving the confirmation of the identity of a body retrieved from water, which was subsequently returned to the institute by its relatives for identification. It also encompasses cases involving maternal half-siblings, complex pedigrees with paternal half-aunts, and others. In each scenario, the total LR was estimated by combining the LR of autosomal STRs (LR AS-STR) with the LR of X-STRs (LR X-STR), utilizing the population frequency database specific to Colombia. Additionally, the LRs of X-STRs obtained using population frequency data from Colombia were compared with those from Mexico. The data derived from both the Mexican and Colombian populations exhibited a high degree of similarity. The collective LR values substantiate the efficacy of X-STR markers, particularly in resolving cases of maternal half-siblings. The analysis underscores the robust informativeness of the 12 X-STR loci examined in the context of forensic testing

Identifying distant relatives using benchtop-scale sequencing

The genetic component of forensic genetic genealogy (FGG) is an estimate of kinship, often conducted at genome scales between a great number of individuals. The promise of FGG is substantial: in concert with genealogical records and other nongenetic information, it can indirectly identify a person of interest. A downside of FGG is cost, as it is currently expensive and requires chemistries uncommon to forensic genetic laboratories (microarrays and high throughput sequencing). The more common benchtop sequencers can be coupled with a targeted PCR assay to conduct FGG, though such approaches have limited resolution for kinship. This study evaluates low-pass sequencing, an alternative strategy that is accessible to benchtop sequencers and can produce resolutions comparable to high-pass sequencing. Samples from a three-generation pedigree were augmented to include up to 7th degree relatives (using whole genome pedigree simulations) and the ability to recover the true kinship coefficient was assessed using algorithms qualitatively similar to those found in GEDmatch. We show that up to 7th degree relatives can be reliably inferred from 1 × whole genome sequencing obtainable from desktop sequencers.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

BackgroundMassively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses.Methods and resultsDNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened.ConclusionsAlthough the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

ORGANIZATIONAL PRINCIPLES OF THE USE OF FORENSIC DNA ANALYSIS IN THE INVESTIGATION OF CRIMINAL OFFENSES

This article studies organizational principles of forensic DNA analysis as the area of practical activities in the field of detection and investigation of criminal offenses. The author defined the list of organizational principles of forensic DNA analysis, disclosed their content, and determined the conditions for implementation in Ukraine. It was established that these principles are: legality, that is, strict compliance with the requirements of the law at all stages of collecting and researching biological material, using and storing genomic information; an exclusively procedural form of using DNA analysis in criminal proceedings; proper functioning of forensic accounting of human genomic information; formation of DNA analysis as a separate section of forensic technology; its organizational and technical support, which primarily involves the accreditation of forensic genetics laboratories according to the European quality standard and compliance with the conditions regarding technical equipment and personnel support. It is substantiated that the implementation of these principles necessitates the need to take a number of regulatory and organizational measures at the national level to ensure at least the minimum conditions for the effective use of forensic DNA analysis technologies, namely: to improve legal norms at least in the aspect of regulation of procedures for obtaining biological material, management of DNA databases and protection of genomic information; determine exclusively the procedural form of using DNA analysis in the criminal process, regulating, in addition to molecular genetic examination, verification studies; to intensify scientific research in the field of criminalistics in the direction of DNA analysis and to introduce relevant topics into educational programs for the training of lawyers and law enforcement officers; to coordinate the work of various DNA analysis laboratories in the context of their accreditation, technical equipment with compatible equipment and personnel training.

Introducing the New Editorial Team

Introducing the New Editorial Team

Simplified COI barcoding of blow, flesh, and scuttle flies encountered in medicolegal investigations.

Accurate insect identification is critical to the estimation of time of colonization (TOC) and post-mortem interval (PMI) in medicolegal death investigations. DNA testing is advantageous because it enables the identification of immature specimens that may not be identified based on morphology alone. We describe here a simplified DNA barcoding method for identifying relevant species that may be implemented by forensic genetics laboratories. A cytochrome oxidase (COI) fragment is analyzed after PCR amplification with a single primer set. The method is effective for many species commonly encountered in death investigations in the USA: members of blowfly genera Calliphora, Chrysomya, Cochliomyia, Lucilia, and Phormia; members of the flesh fly genera Blaesoxipha, Oxysarcodexia, Ravinia, and Sarcophaga; and the scuttle fly Megaselia scalaris. We tested the method on specimens with verified identifications and used it to build a collection of reference sequences from specimens collected in Harris County, Texas. We show here the correct identification of larvae, pupae, and pupal exuviae from the medicolegal casework.

A comparison of five DNA extraction methods from degraded human skeletal remains

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

Aportaciones y avances de la genética forense en los sucesos con víctimas múltiples

Disaster victim identification is crucial for humanitarian and legal reasons. Forensic genetics plays an important role in these situations which often become a challenge for the different professionals involved due to their complexity. The establishment of guidelines and recommendations makes it easier to follow standardized protocols that make it possible to guarantee the reliability of the identification final result. Likewise, advances in forensic genetics contribute to speeding up the response, providing new analysis strategies and bioinformatic tools. This article aims to provide an overview of how forensic genetics and its advances can contribute in these situations, as well as some keys to understanding the work of forensic genetics laboratories in the identification of corpses in events with multiple victims.

Contributions and advances of forensic genetics in mass fatality incidents

Disaster victim identification is crucial for humanitarian and legal reasons. Forensic genetics plays an important role in these situations which often become a challenge for the different professionals involved due to their complexity. The establishment of guidelines and recommendations makes it easier to follow standardised protocols that make it possible to guarantee the reliability of the identification final result. Likewise, advances in forensic genetics contribute to speeding up the response, providing new analysis strategies and bioinformatic tools. This article aims to provide an overview of how forensic genetics and its advances can contribute in these situations, as well as some keys to understanding the work of forensic genetics laboratories in the identification of corpses in events with multiple victims.

Evaluating the sensitivity of presumptive and confirmatory tests for body fluids

Most exhibits received in the forensic genetics laboratory of Ras Al Khaimah (United Arab Emirates) are swabs or clothing with stains suspected of being either blood, semen or saliva; swabs from touch DNA are also reasonably common. Routine practice has been to use presumptive tests to characterize the materials before DNA extraction. In this study we evaluated the presumptive methods currently used for blood (Hemastix® and Kastle-Meyer), semen (Phosphatesmo KM) and saliva (Phadebas®) in comparison to the confirmatory tests OBTI Hexagon and RSID™ Blood, RSID™ Semen and RSID Saliva™. Results from this study showed that, as expected, presumptive tests were generally more sensitive in detecting body-fluids than confirmatory tests. The presumptive tests were at least two-fold more sensitive than the RSID™ Blood, Semen and Saliva confirmatory tests. However, the OBTI Hexagon test showed comparable sensitivity to Kastle-Meyer and Hemastix® for the detection of blood; this test utilizes an anti-hemoglobin antibody, which enables the high level of sensitivity. DNA was extracted from different dilutions and quantified using real-time PCR with the Quantifiler™ Human kit. Except for the RSID™-Blood and Saliva, the limit of detection for the tests was at dilutions where recovery of sufficient DNA for STR analysis was not likely.

The extent of STR chimerism in different biological samples following bone marrow transplantation: A case report

Biological samples recovered from recipients of allogeneic haematopoietic stem cell transplant (HSCT) contain genetic material from both donor and him/herself. This chimeric condition can greatly complicate analysis of a DNA evidence and undermine its power of discrimination, as the specimens could be mistakenly identified as a mixed sample when in fact it originated from a single person. Profiling reference samples could help clarify the profile anomalies, however, the degree of mixture between host’s and donor’s genetic materials has been reported to vary depending on the tissue from which DNA were obtained. As a result, knowledge to select appropriate sources of reference samples that would most likely reveal the person’s pre- and post-transplant alleles becomes necessary to minimise any possible misunderstanding during the subsequent DNA profile interpretation and comparison. This work investigated the extent of chimerism present in different types of biological samples collected from an individual who had undergone a bone marrow transplant as a child (post-transplant interval, PTI >28 years). DNA profiles from buccal cells, saliva, hair roots, and fingernail clippings were generated using AmpFℓSTR™ Identifiler™ Plus/Direct PCR Amplification Kit. The donor’s STR profile was used to identify donor- and host-specific alleles (DSA and HSA, respectively), and relative donor chimerism (%Ch) of each marker was calculated. Results showed that the saliva sample contained the highest level of chimerism, with complete profile of donor being detected and the mean %Ch was 34.0 ± 4.5. Four out of eight DSA dropped out from the buccal cell profile, and the mean %Ch was 24.2 ± 1.7. Hair roots and fingernail clippings yielded single source profiles and only host’s original alleles were present. Except for the fingernail results, these observations agreed with many of the previous studies, which further demonstrated the need for raising awareness among forensic genetic laboratories regarding the type of reference samples that should be collected in future cases.

Test of chlorine wipes for efficient removal of DNA from forensic genetics laboratories

Sodium hypochlorite is an efficient reagent for removal of unwanted DNA from laboratory surfaces. Here, we tested two different chlorine wipes and compared their performance to a 0.9–1.8% hypochlorite solution. WipeClean Chlorine Disinfection wipes contain > 0.1 g sodium hypochlorite/kg, whereas WetWipe Chlorine Desinfection wipes contain > 1000 ppm active chlorine. Clean surfaces were contaminated with 10 µL 0.5 ng/µL of massively parallel sequencing libraries. The DNA was dried and left for 45 min before any treatment. The surfaces were cleaned using either 1) a 0.9–1.8% hypochlorite solution and clean wipes, 2) a WipeClean wipe, 3) a WetWipe, or 4) the surface was not cleaned. All experiments were repeated three times. Subsequently, the surfaces were swabbed using cotton swabs. DNA was extracted from the swabs and the DNA concentrations were determined in quadruplicates by real-time PCR. This protocol was repeated after the soft plastic wrapping around the wipes were left open or closed for several weeks. The results showed that the WipeClean wipes efficiently removed DNA for up to four weeks after the box with the wipes were opened, whereas the WetWipe wipes dried faster and gradually lost their cleaning effect.

A Monte Carlo simulation and sensitivity cost benefit analysis for use of nylon 4N6FLOQSwabs®

Forensic genetic laboratories are challenged with implementing innovation even if the benefits to operational performance are well demonstrated often because of internal budget constraints. A prospective cost–benefit analysis (CBA) could support justification for an increased budget by effectively demonstrating in a system-based approach the relatively small cost of increasing a laboratory budget can substantially reduce costs to society (both qualitatively and monetarily). A Monte Carlo simulation and sensitivity CBA was performed using a more expensive swab (i.e., nylon 4N6FLOQSwabs®) compared with a less costly cotton swab. Ranges of input values and tangible and intangible benefits were considered. The outcome is that the relatively small increased cost of using a nylon swab pales compared with the potential tangible and intangible benefits to the overall system. This approach provides a sounder basis for requesting additional funds to support implementation of technologies and better approximates realistic situations while accommodating uncertainty of input values.

Application of 3D printing in assessment and demonstration of stab injuries

In stabbing related fatalities, the forensic pathologist has to assess the direction of wound track (thus, the direction of the stabbing) and the weapon’s possible characteristics by examining the stab wound. The determination of these characteristics can be made only with a high level of uncertainty, and the precise direction of the stabbing is often difficult to assess if only soft tissues are injured. Previously reported techniques used for the assessment of these wound characteristics have substantial limitations. This manuscript presents a method using today’s easily accessible three-dimensional (3D) printing technology for blade-wound comparison and wound track determination. Scanning and 3D printing of knives is a useful method to identify weapons and determine the precise stabbing direction in a stabbing incident without compromising the trace evidence or the autopsy results. Ballistic gel experiment, and dynamic stabbing test experiments prove the method can be applied in safety, without compromising the autopsy results. Identification of the exact knife is not possible with complete certainty but excluding certain knives will decrease the number of necessary DNA examinations, hence it can lower the burden on forensic genetic laboratories. The method addresses many of the shortcomings of previously used methods of probe insertion or post-mortem CT. Insertion of the printed knife into the wound gives a good visual demonstration of the stabbing direction, thus easing the forensic reconstruction of the stabbing incident. After combining the 3D printing with photogrammetry, the achieved 3D visualization is useful for courtroom demonstration and educational purposes.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Review of the Forensic Applicability of Biostatistical Methods for Inferring Ancestry from Autosomal Genetic Markers.

The inference of ancestry has become a part of the services many forensic genetic laboratories provide. Interest in ancestry may be to provide investigative leads or identify the region of origin in cases of unidentified missing persons. There exist many biostatistical methods developed for the study of population structure in the area of population genetics. However, the challenges and questions are slightly different in the context of forensic genetics, where the origin of a specific sample is of interest compared to the understanding of population histories and genealogies. In this paper, the methodologies for modelling population admixture and inferring ancestral populations are reviewed with a focus on their strengths and weaknesses in relation to ancestry inference in the forensic context.

Forensic DNA Analysis: Development State and Prospects in Ukraine

This research purpose is to summarize information on the main directions of using DNA analysis in the practice of detection and investigation of crimes used in different countries, consider possibilities of these areas and the state of their implementation in domestic investigative and forensic expert practice and formulate the author’s vision of prospects for the further development of theoretical and applied aspects of forensic DNA research in Ukraine.
 Traditional DNA profiling methods based on STR locus analysis and mtDNA SNP research are described, information on the application of the latest technologies for searching relatives by DNA profile bases and determining the age and biogeographical origin of human DNA, as well as methods of mass parallel DNA sequencing and performing rapid DNA profile tests. It was noted that main obstacle to the further development of DNA analysis in Ukraine is the lack of proper legal regulation that hinders formation of effective national database of DNA profiles. Attention is drawn to the obvious lack of scientific support for DNA research in Ukraine.
 Ways to solve existing issues in this area are proposed, in particular: promote relevant knowledge among law enforcement officers to minimize typical errors in handling of traces of biological origin; make changes in the criminal procedural legislation of Ukraine for the purpose of proper adjustment of issues of sampling of biological samples and carrying out rapid analyzes on a DNA profile; take measures to expand the tool base and real capabilities of forensic genetics laboratories.

First experiences with the Spectrum Compact CE System

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.