DNA Analysis Methods Research Articles

Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit

The YFiler Platinum Casework PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 38 male-specific, Y-chromosome Short Tandem Repeat (YSTR) markers (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS549, DYS593, DYS645, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS557, DYS522, DYS456, DYS390, DYS438, DYS392, DYS518, DYS444, DYS533, DYS570, DYS437, DYS385, DYS449, DYS643, DYS596, DYS393, DYS439, DYS481, DYF387S1, DYS527, DYS447), three insertion/deletion polymorphic markers (Yindels: rs771783753, rs759551978, rs199815934), and an internal quality control (IQC) system. When compared to the YFiler Platinum PCR Amplification kit for database samples, YFiler Platinum Casework kit was developed to include an improved Primer Mix incorporating a brighter TED dye, an updated internal quality control system, better resolution of large DNA fragments in Applied Biosystems POP-4 Polymer, and reduced female DNA cross-reactivity. Here, we report the results of the developmental validation study which followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, population concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

The role of DNA in forensic science: A comprehensive review

Forensic genetics, leveraging molecular tools and scientific applications, has witnessed significant advancements in DNA analysis over the last three decades. These progressions have enhanced the discrimination power, speed, and sensitivity of DNA profiling methods, enabling the analysis of challenging samples. This article explores the significance of forensic genetics in criminal investigations, traces the historical evolution of DNA analysis techniques, and presents recent developments in the field. The article aims to provide a comprehensive understanding of the crucial role of forensic genetics in criminal investigations and sheds light on the latest trends and breakthroughs in this area. The evolution of DNA typing from ABO blood typing to the current standard of short tandem repeat (STR) analysis is discussed, along with alternative DNA analysis methods, such as Y-chromosome analysis and single nucleotide polymorphism (SNP) typing. Massively parallel sequencing (MPS) represents a groundbreaking advancement, enabling whole genome sequencing and addressing complex cases. The article also covers recent innovations, including DNA methylation analysis, body fluid identification, forensic DNA phenotyping, and genetic genealogy, highlighting their potential benefits in forensic investigations. Despite these advancements, standard STR profiling remains the gold standard due to its established protocols and databases. Ethical considerations regarding data privacy and cost implications are crucial as these technologies continue to progress in their pursuit of justice.

Insight into Trophic Interactions of Spiders in Olive Groves with Integrated and Ecological Pest Management Using DNA Metabarcoding

Spiders act as one of the major predators among arthropods in agroecosystems and are crucial to the control of pest densities. As they are almost exclusively predators, they have developed a number of hunting strategies, which have consequently impacted their complex trophic interactions. The development of molecular biology methods for environmental DNA analysis has made it easier to analyze such complex trophic networks. In order to ascertain the trophic interactions of spiders, a sampling of spider species was carried out in two olive groves in Zadar County, Croatia, under integrated and ecological pest management. To construct the trophic networks of spiders, we performed DNA metabarcoding. The combined general and spider-excluding primers were able to recognize prey from 12 distinct orders in the guts of the 57 spiders. According to our results, spiders have a complex trophic network that exhibits seasonal and site-specific variations. The results obtained from both sites having different pest management also confirmed that spiders consumed phytophagous insects in the highest ratio, including some important pests, in comparison to other prey and that management and guild type had an impact on the predation of phytophagous insects.

A study of genetic profiles of grape plants preserved under the name of Dagestan variety ‘Khatmi’

Background. Traditionally, the description of grape varieties is a task of ampelographic studies. However, several different grape cultivars have similar phenotypic traits. Molecular genetic characterization is the most accurate tool for cultivar identification. The development of DNA fingerprinting of varieties is the first step in this direction. An extensive database of DNA profiles of grape genotypes makes it possible to determine the varietal affiliation of unknown forms, confirm or refute the varietal correspondence of planting material. ‘Khatmi’ is an autochthonous grape variety from Dagestan. The profile of ‘Khatmi’ is presented in the VIVC international database of DNA fingerprints for grape varieties. However, an application of DNA analysis methods in grape variety studies has determined that several ancient varieties were cultivated under one name, while for others a certain variability of genotypes was observed. The objectives of the work were to study samples of ‘Khatmi’ plants from different places of growth in Dagestan by standard microsatellite loci used for grape genotyping, to assess the level of genetic similarity of the samples, and to refine the DNA profile of ‘Khatmi’. Materials and methods. Molecular genetic study was carried out on 10 samples from different ‘Khatmi’ populations. The material was picked from the collection sites of Dagestan breeding experimental station of viticulture and vegetable growing and the Dagestan Experiment Station of VIR, as well as from production plantations. DNA was extracted from herbarium specimens of young grape shoot tips by the CTAB method. The samples were genotyped by polymerase chain reaction using a standard set of primers for 9 microsatellite markers VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79 recommended by the International Organization of Grapes and Wine (OIV) for grapevine DNA fingerprinting. Amplification products were separated, and their sizes were assessed using capillary electrophoresis on an ABI Prism 3130 genetic analyzer. Results. Genotyping was done for 10 samples of grapes growing in Dagestan under the name ‘Khatmi’, including samples from different collections and places of industrial cultivation, as well as clonal variations of this variety and putative clonal variations. The two base pair differences within one of the loci distinguished the DNA profiles of the analyzed samples from that of ‘Khatmi’ presented in the international grape varieties database VIVC. It was determined that the sample under the name ‘Khatmi krupnoyagodnyi’ is closely related to ‘Khatmi’ variety by its genotype, but probably represents a clonal variation of ‘Koz uzyum’, another local variety of Dagestan. Conclusion. The DNA profile of the local Dagestan grape variety ‘Khatmi’ has been refined.

Environmental RNA can distinguish life stages in amphibian populations.

Applications of environmental DNA (eDNA) analysis methods for biomonitoring have grown exponentially over the last decade and provide a wealth of new information on the distribution of species. However, eDNA methods have limited application for estimating population-level metrics. Environmental RNA (eRNA) has the potential to address ecological questions by gathering population demographic information from environmental media but may be challenging to detect and analyze. We developed gene-specific eRNA assays targeting keratin-associated genes in two focal species, American bullfrogs (Lithobates catesbeianus) and tiger salamanders (Ambystoma mavortium) to answer an important question in amphibian management: whether species detections represent breeding populations versus transitory adults. We performed an extensive laboratory validation with amphibians housed across development stages, where we collected 95 and 127 environmental samples for bullfrogs and salamanders, respectively. Both assays were highly specific to the larval stage and amplified with high sensitivity (90% in bullfrog and 88.4% in tiger salamander samples). We then applied our validated assays to multiple natural systems. When larvae were present, we found 74.1% overall detection in bullfrog field samples and 70.8% and 48.5% overall detection in field samples from ponds with A. macrodactylum and A. californiense larvae, correlating with eDNA detection rates. When only adults were present, we did not detect larvae-specific eRNA in A. macrodactylum ponds, despite high eDNA detection rates. Although much work is ahead for optimizing assay design, sampling and filtering methods, we demonstrate that eRNA can successfully be used to discern life stages with direct application for ecology and conservation management.

Benthic invertebrate biodiversity enhancement with reef cubes®, evidenced by environmental DNA analysis of sediment samples

Nature inclusive design (NID) is an “eco-friendly” approach to the construction of manmade infrastructure. reef cubes® are patented construction products designed to be used as NID options for marine developments. Artificial reefs can be made with reef cubes® when used either as replacements of or additions to conventional marine manmade structures in marine construction projects. The first long-term, marine research station with reef cubes® is in Torbay, Devon, UK. A goal of the installation, to enhance biodiversity, is assessed here in regards to the benthic invertebrate community, with a novel method of sedimentary environmental DNA (eDNA) analysis. There are two primary aims targeted in this study: firstly to use sedimentary eDNA metabarcoding to assess if the enhancement of invertebrate biodiversity has been achieved and secondly to assess if the reef cubes have a negative impact on the infaunal diversity of exposed sediments in their vicinity. Sediment samples were taken in and away from, the reef cube® test site, in periods before and after their installation. Genetic material in the samples was amplified, sequenced, and amplicon sequence variants (ASVs) assigned to taxonomic ranks. Invertebrate metazoan biodiversity was analysed in terms of taxonomic richness, taxonomic diversity, genetic diversity of ASVs per taxa and species richness of: orders, functional feeding groups and positional lifestyle groups (infauna, epifauna or plankton). A significantly higher biodiversity of invertebrates was found at the reef sites across all measures. Benthic epifauna, suspension feeders and carnivore taxa in particular exhibited higher diversity. Mussels, barnacles, anemones, hydrozoans and copepods were the most distinguishing taxa. Benthic infauna were similarly diverse inside and around the reef cubes® to surrounding sediments. The results suggest that at least for the reef cubes® sampled here the goal of enhancing invertebrate biodiversity at the test site has been achieved. There was no negative effect found on the infaunal diversity in exposed sediments inside and beside the reef cubes®. Application of eDNA analyses to sediment samples represents a promising and efficient method for assessing such ecological goals with the installation of NIDs and artificial reefs.

Development of a multiplex panel with 36 insertion/deletion markers (InDel) for individual identification.

In recent years, the insertion/deletion (InDel) polymorphism has become a preferred genetic marker in forensic genetics due to its low mutation rates and small amplicon sizes. In this study, a 36-InDelplex identification panel, consisting of autosomal 34 InDel loci, 1 Y InDel locus, and amelogenin, was developed, and gene frequencies in the Turkish population were determined. The loci of the InDel panel with global minimum allele frequencies (MAF) ≥ 0.4 were selected from the 1000 Genomes Project Phase 3 data. The amplicon sizes of the loci were designed in the range of 69-252 bp. In the validation study of the developed panel, analysis threshold, dynamic range, sensitivity, stochastic threshold, inhibitor tolerance, and reproducibility parameters were studied by following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The sensitivity studies indicated that complete and reliable InDel profiles could be obtained with 0.25 ng of DNA. A population study was evaluated using 250 samples from Turkey. The mean observed heterozygosity ratio (Ho) of all loci was 0.48. The combined discrimination power (CPD) is 0.999999999990867 and the combined exclusion probability (CPE) was 0.9930. The population comparison was also made using Turkish and the five major populations from the 1000 Genomes Phase 3 populations' data (Africa, Europe, East Asia, South Asia, and America). In conclusion, the results showed that the 36-InDelplex panel is a reliable, sensitive, and accurate system that is suitable for human identification and population genetics purposes.

The potential of microsatellite markers on the identification of various ethnic alleles in Indonesia

Indonesia is an archipelagic country with a high level of population diversity such as religion, ethnicity, culture and also genetic diversity. Environmental factors also have an impact on the genetic variation and diversity of the population, including high mobility and migration triggering the tendency for inter-ethnic marriages to occur. This environmental factor is one of the reasons for research based on DNA analysis on ethnicity in Indonesia for the purpose of forensic identification. One of the DNA analyzes for forensic identification is the microsatellite DNA analysis method with a high sensitivity level.

Development and application of a multiplex PCR system for forensic salivary identification.

In forensics, accurate identification of the origin of body fluids is essential for reconstructing a crime scene or presenting strong evidence in court. Microorganisms have demonstrated great potential in body fluid identification. We developed a multiplex PCR system for forensic salivary identification, which contains five types of bacteria:Streptococcus salivarius, Neisseria subflava, Streptococcus. mutans, Bacteroides thetaiotaomicron, and Bacteroides. uniformis. And the validated studies were carried out following the validation guidelines for DNA analysis methods developed by the Scientific Working Group on DNA Analysis Methods (SWGDAM), which included tests for sensitivity, species specificity, repeatability, stability, and mixed samples, trace samples, case samples, and a population study. Our result depicted that the lowest detection limit of the system was 0.01ng template DNA. Moreover, the corresponding bacteria can still be detected when the amount of saliva input is low to 0.1 μL for DNA extraction. In addition, the target bacteria were not detected in the DNA of human, seven common animals, and seven bacteria DNAand in nine other body fluid samples (skin, semen, blood, menstrual blood, nasal mucus, sweat, tears, urine, and vaginal secretions). Six common inhibitors such as indigo, EDTA, hemoglobin, calcium ions,alcohol and humic acid were well tolerated by the system. What is more, the salivary identification system recognized the saliva component in all mixed samples and simulated case samples. Among 400 unrelated individuals from the Chinese Han population analyzed by this novel system, the detection rates of N. subflava, S. salivarius, and S. mutans were 97.75%, 70.75%, and 19.75%, respectively, with 100% identification of saliva. In conclusion, the salivary identification system has good sensitivity, specificity, stability, and accuracy, which can be a new effective tool for saliva identification.

A Robust Test System for DNA Identification of Raccoon Dogs

To establish a system for DNA identification of biological samples of the raccoon dog (Nyctereutes procyonoides), we carried out a study of polymorphism of 39 microsatellite loci and three sex loci specific to various species of the Canidae family. This resulted in the development of the NPlex test system, which includes 14 autosomal STRs and two sex loci. The test system has been validated for use in forensic identification of racoon dogs based on biological traces (such as traces of blood and secretions, fragments of muscle tissue or bones, dermis and hair, etc.) found at crime scenes, as well as for establishing biological relationship of animals of this species. Validation efforts were performed in accordance with the protocol of the Scientific Working Group on DNA Analysis Methods.

A systematic review of DNA-based methods in authentication of game and less common meat species.

Despite the numerous studies on food safety and authenticity, especially for meat and meat products, not enough studies have been conducted focusing exclusively on game species and other unusual meat animals. As a result of the European horse scandal, the horse is currently the target of many meat authenticity studies. With this review, we aim to present various DNA-based methods that have been used by researchers to identify, detect, and quantify game, uncommon meat animals, and wildlife species. Polymerase chain reaction (PCR) is considered the standard method for DNA analysis in meat authenticity testing. However, in this paper, we present several other methods that may or may not involve the PCR technique. For this purpose, we systematically reviewed 131 articles selected according to various criteria such as target animal species, method of analysis, year of publication, and so forth. The result of our study shows the most studied game and uncommon meat species, PCR- and non-PCR-based methods for game meat analysis, and DNA-based methods in wildlife conservation. With this study, researchers can find detailed information about frequent game species used as adulterants for regular meat products and the DNA-based techniques to identify them.

Developmental Validation of the Illumina Infinium Assay Using the Global Screening Array on the iScan System for Use in Forensic Laboratories

Microarray processing, which interrogates hundreds of thousands of single nucleotide polymorphisms (SNPs) across the human genome, has recently gained traction in forensics due to its use in forensic genetic genealogy, which is based on analysis using SNPs to compare distant relatives in publicly curated databases for the purposes of developing investigative leads or identifying human remains. To date, there has been no published developmental validation of microarray processing using the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for DNA Analysis Methods and Federal Bureau of Investigation Quality Assurance Standards. Validation of these methods are warranted to identify samples suitable for microarray analysis and to assess the quality of the data obtained before upload to genealogical databases. In this study, we validated the Global Screening Array for use in forensic investigations according to SWGDAM guidelines, including the following studies: precision and accuracy, sensitivity, contamination, degradation, species specificity, mock case-type samples, mixtures, repeatability and reproducibility, and stability. Results indicated accurate genotype calls with SNP call rates >95% at DNA input as low as 0.20 ng. In addition to SNP call rate, we developed interpretation thresholds for signal intensity and heterozygosity to allow for sample quality assessment and identification of highly degraded and/or non-human DNA. This study demonstrates that high-quality data can be generated from multiple sample types, including mock forensic evidence that simulated the challenges that are often encountered in forensic cold cases.

Predicting Cherax quadricarinatus Habitat Distribution Patterns Through the Usage of GIS and eDNA Analysis in Terengganu, Malaysia

Cherax quadricarinatus (von Martens, 1868) is a highly invasive species that is resilient and highly adaptable to environmental conditions in new locations. Its presence brings along ecosystem imbalances and causes socioeconomic losses in invaded areas. Due to the destructive behaviour, it is important to conduct preliminary assessment methods and scientific studies especially on its behavioural and dispersal patterns. Thus, this study aims to determine the habitat distribution patterns of C. quadricarinatusbased on the methods of GIS and environmental DNA (eDNA) analysis. The study was done at Felda Tenang, Terengganu, Malaysia. Based on the geographical features and climate conditions of the study area, we theorized that there are fewer and older C. quadricarinatuspresent at higher areas but more and younger C. quadricarinatus at lower areas. We found that 91.67% of the study area to be invaded, thus imploring the need for future mitigation plans to curb their dispersal into new areas. Future studies should also be done to determine the habitat distribution patterns of C. quadricarinatus in other areas.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

方法変更後のミトコンドリアDNA検査法の検証

The Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines for DNA analysis method indicate that when material modifications are made to an existing procedure that may affect the results of the analysis, the concordance between the results before and after the modifications should be confirmed. In this study, we modified our conventional procedure for mitochondrial DNA (mtDNA) analysis by changing the mtDNA quantification and sequence product purification methods along with and without changing the version of the data collection software and/or sequencing analysis software. We performed mtDNA analysis using the four modified procedures and ensured the concordance between the results obtained before and after the modifications. The sensitivity was also confirmed to be comparable among the conventional and all the modified procedures. Therefore, we concluded that the modifications can be adopted in the procedures for our mtDNA analysis.

Synergistic Distributed Thermal Regulation for On-CMOS High-Throughput Multimodal Amperometric DNA-Array Analysis

Accurate temperature regulation is critical for amperometric DNA analysis to achieve high fidelity, reliability, and throughput. In this work, a 9×6 cell array of mixed-signal CMOS distributed temperature regulators for on-CMOS multi-modal amperometric DNA analysis is presented. Three DNA analysis methods are supported, including constant potential amperometry (CPA), cyclic voltammetry (CV), and impedance spectroscopy (IS). In-cell heating and temperature sensing elements are implemented in standard CMOS technology without post-processing. Using proportional-integral-derivative (PID) control, the local temperature can be regulated to within ± 0.5∘C of any desired value between 20∘C and 90∘C. To allow the in-cell integration of independent PID control, a new mixed-signal design is proposed, where the two computationally intensive operations in the PID algorithm, multiplication, and subtraction, are performed by an in-cell dual-slope multiplying ADC, resulting in a small area and low power consumption. Over 95% of the circuit blocks are synergistically shared among the four operating modes, including CPA, CV, IS, and the proposed temperature regulation mode. A 3mm×3mm CMOS prototype fabricated in a 0.13μm CMOS technology has been fully experimentally characterized. The proposed distributed temperature regulation design and the mixed-signal PID implementation can be applied to a wide range of sensory and other applications.

BILL S-231: The Ethics of Familial and Genetic Genealogical Searching in Criminal Investigations

Recent breakthroughs in criminal investigations, especially of high-profile cold cases, have helped to consolidate the role of DNA analysis in investigative contexts. Consequently, some jurisdictions are looking to expand DNA collection and analysis methods. In Canada, legislation has been proposed to expand the National DNA Databank (NDDB) and to allow familial searching in criminal and forensic investigations. This article outlines the ethical implications of the proposed legislation and, more broadly, of genealogical methods already in use that operate outside the NDDB and rely heavily on for-profit and consumer DNA services. Current DNA analysis within the criminal justice system is heavily regulated and provides important protections not only for individuals but also for genetic relatives whose biometric data is indirectly implicated. In contrast, familial searching poses risks for offender privacy as well as for their relatives. Additionally, the expanding practice of genetic genealogical searching relies on unregulated commercial products that use different technology to expose highly detailed genetic information. This technology falls short of rigorous investigational standards and poses significant problems for informed consent. We conclude that expanding DNA collection within the NDDB to include familial searching risks exacerbating existing systemic bias and that genetic genealogical searching outside of the NDDB is incompatible with existing Canadian legislation that safeguards privacy, genetic non-discrimination, and fundamental rights and freedoms.

Development and validation of a new multiplex for upgrading Y-STRs population databases from 12 to 23 markers and its forensic casework application

Y chromosomal short tandem repeats (Y-STRs) are used in forensic investigations as a useful complementary tool to autosomal markers. The ongoing development of new kits with an increasing number of markers makes it necessary to update populations typed in the Y-STR Haplotype Reference Database to reach at least 23 Y-STRs. A novel Y-STR multiplex panel was developed to offer a cost-efficient alternative to update Y-STR haplotypes from 12 to 23 loci. This panel includes the eleven markers, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS576, DYS481, DYS549, DYS533, DYS570 and DYS643, as well as DYS385a/b for traceability purpose. Developmental validation of this panel was conducted following the recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM), showing high sensitivity, tolerance to common inhibitors as well as high species specificity. It was efficient for degraded DNA samples and for detection of male mixtures. When applying it for extending the current data of the Ibiza population, both the discrimination capacity and the haplotype diversity increased from 0.5952 to 0.9048 and from 0.9808 to 0.9977, respectively. Together, the study demonstrates the suitability of this panel in forensic casework.

A new fluorescence labeling method for molecular analysis of double-stranded DNA

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Developmental validation of the EZ1&2™ DNA Investigator® Kit on the EZ2® Connect Fx

The EZ1&2 DNA Investigator Kit allows automated purification of total DNA from forensic and human identity samples on the EZ2 Connect Fx instrument. Its proven magnetic-bead technology provides high-quality DNA, suitable for direct use in sensitive downstream applications such as quantitative PCR, STR analysis and NGS. The purified DNA obtained is free of proteins, nucleases and inhibitors. In this study, the performance of the EZ1&2 DNA Investigator Kit on the EZ2 Connect Fx was evaluated using typical casework sample types and conditions commonly encountered in forensic and parentage laboratories. Wherever applicable, this validation study followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) and the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM).