Field Of Forensic Genetics Research Articles

Comparative analysis of SNaPshot and massively parallel sequencing for body fluid-specific DNA methylation markers.

The identification of tissue-specific differentially methylated regions has significantly contributed to the field of forensic genetics, particularly in body fluid identification crucial for linking evidence to crimes. Among the various approaches to analyzing DNA methylation, the SNaPshot assay has been popularly studied in numerous researches. However, there is a growing interest in exploring alternative methods such as the use of massively parallel sequencing (MPS), which can process a large number of samples simultaneously. This study compares SNaPshot and MPS multiplex assays using nine cytosine-phosphate-guanine markers for body fluid identification. As a result of analyzing 112 samples, including blood, saliva, vaginal fluid, menstrual blood, and semen, both methods demonstrated high sensitivity and specificity, indicating their reliability in forensic investigations. A total of 92.0% samples were correctly identified by both methods. Although both methods accurately identified all blood, saliva, and semen samples, some vaginal fluid samples showed unexpected methylation signals at nontarget loci in addition to the target loci. In the case of menstrual blood samples, due to their complexity, independent typing criteria were applied, and successful menstrual blood typing was possible, whereas a few samples showed profiles similar to vaginal fluid. The MPS method worked better in vaginal fluid samples, and the SNaPshot method performed better in menstrual blood samples. This study offers valuable insights into body fluid identification based on the characteristics of the SNaPshot and MPS methods, which may help in more efficient forensic applications.

Artificial Intelligence and Forensic Genetics: Current Applications and Future Perspectives

The term artificial intelligence (AI) was coined in the 1950s and it has successfully made its way into different fields of medicine. Forensic sciences and AI are increasingly intersecting fields that hold tremendous potential for solving complex criminal investigations. Considering the great evolution in the technologies applied to forensic genetics, this literature review aims to explore the existing body of research that investigates the application of AI in the field of forensic genetics. Scopus and Web of Science were searched: after an accurate evaluation, 12 articles were included in the present systematic review. The application of AI in the field of forensic genetics has predominantly focused on two aspects. Firstly, several studies have investigated the use of AI in haplogroup analysis to enhance and expedite the classification process of DNA samples. Secondly, other research groups have utilized AI to analyze short tandem repeat (STR) profiles, thereby minimizing the risk of misinterpretation. While AI has proven to be highly useful in forensic genetics, further improvements are needed before using these applications in real cases. The main challenge lies in the communication gap between forensic experts: as AI continues to advance, the collaboration between forensic sciences and AI presents immense potential for transforming investigative practices, enabling quicker and more precise case resolutions.

The present and future of forensic genetics

In this article, we discuss the current state and future directions in the field of forensic genetics. The DNA analysis of biological traces found at a crime scene, which was first used in a criminal investigation in 1987, did revolutionize forensic science. Over the past three decades, significant advances have been made in the recognition capacity, speed, and sensitivity of DNA profiling methods, as well as in their capability of typing increasingly complex patterns. Creation of DNA databases of criminals and crime scenes, as well as population allele frequencies, allows suspects to be identified from crime scene samples and DNA evidence to be statistically processed to verify its reliability. At present, it has become possible to identify even single cells left at a crime scene and to successfully analyze ancient human remains. Forensic DNA profiling can be used to identify not only individuals known to the investigating authorities. Experts are increasingly applying new genetic markers that can expand the scope of DNA profiling methods. Modern developments enable extraction of new types of forensically significant information from biological traces, e.g., using molecular approaches to searching for individuals previously unknown to investigators. New methods have been proposed to identify the relationship between the donors of forensic samples and the crimes committed. Modern advances in the decoding of the human genome, as well as the availability of genome-wide analysis and sequencing techniques, pave the way for new forensic DNA tools capable of enhancing the quality of forensic science in the near future.

Technical strategy for monozygotic twin discrimination by single-nucleotide variants.

Monozygotic (MZ) twins are theoretically genetically identical. Although they are revealed to accumulate mutations after the zygote splits, discriminating between twin genomes remains a formidable challenge in the field of forensic genetics. Single-nucleotide variants (SNVs) are responsible for a substantial portion of genetic variation, thus potentially serving as promising biomarkers for the identification of MZ twins. In this study, we sequenced the whole genome of a pair of female MZ twins when they were 27 and 33years old to approximately 30 × coverage using peripheral blood on an Illumina NovaSeq 6000 Sequencing System. Potentially discordant SNVs supported by whole-genome sequencing were validated extensively by amplicon-based targeted deep sequencing and Sanger sequencing. In total, we found nine bona fide post-twinning SNVs, all of which were identified in the younger genomes and found in the older genomes. None of the SNVs occurred within coding exons, three of which were observed in introns, supported by whole-exome sequencing results. A double-blind test was employed, and the reliability of MZ twin discrimination by discordant SNVs was endorsed. All SNVs were successfully detected when input DNA amounts decreased to 0.25ng, and reliable detection was limited to seven SNVs below 0.075ng input. This comprehensive analysis confirms that SNVs could serve as cost-effective biomarkers for MZ twin discrimination.

Cracking the code: Can forensic genetics distinguish identical twins? A technical perspective

DNA fingerprinting has revolutionized the field of forensic investigation in recent times. The utility of DNA fingerprinting technology in differentiating between monozygotic twins has always been an area of academic interest for researchers. While traditional chiral fingerprinting effectively differentiates monozygotic twins, situations may arise where the absence of conventional fingerprints at a crime scene necessitates DNA analysis for conclusive identification. Although Short Tandem Repeats (STR) profiling is of not much help in that case, monozygotic twins can still be differentiated using current advancements in the field of forensic genetics. The methods that can help discriminate between identical twins are mitochondrial genome (mtGenome) analysis, Single Nucleotide Polymorphism (SNP) profiling, Epigenetic profiling (DNA methylation profiling), Copy Number Variants (CNV) profiling, and studying Single Nucleotide Variants (SNV) in the genetic material by traditional sequencing methods like sanger sequencing or advanced methods like Whole Genome Sequencing (WGS). In conclusion, these alternative methods may be able to distinguish between monozygotic twins, but they also have drawbacks that must be considered when using them in real-world crime investigations. To establish their accuracy, dependability, and application in forensic situations, more study, validation, and standardization are required. A combination strategy will strengthen the case and lessen the fallacy of individual profiling methods.

Forensic Analysis of Poaching: Wildlife in the Legal Arena

Poaching and the international wildlife trade are growing problems, fueled by a confluence of greed and poverty and managed by ever-more-evolving criminal networks. These actions lead to the loss of biodiversity, which is made worse by changes in habitat and drives many species to the verge of extinction. In this situation, forensic evidence is the cornerstone of investigations into crimes involving both people and animals, and it is essential to securing convictions and criminal prosecutions. The application of forensic methods—especially in the field of forensic genetics—is constantly progressing, giving investigators the instruments they need to address the many problems associated with wildlife-related inquiries.
 This article highlights current developments while examining the evolution of these forensic tools and their useful uses. In addition, it provides examples of actual cases where forensic evidence was crucial in obtaining convictions, paving the way for the eventual application of similar methods to dismantle criminal organizations and safeguard species for the sake of maintaining biodiversity.

Pitfalls and challenges with population assignments of individuals from admixed populations: Applying Genogeographer on Brazilian individuals

The assignment of individuals to a population can be of importance for the identification of mass disaster victims or criminal offenders in the field of forensic genetics. This assignment is based on biostatistical methods that process data of ancestry informative markers (AIMs), which are selected based on large allele frequency differences between the populations of interest.However, population assignments of individuals with an admixed genetic background are challenging. Admixed individuals are genetic mosaics of chromosomal segments from the parental populations, which may lead to ambiguous or no population assignment. This is problematic since admixture events are a substantial part of human history.In this study, we present challenges of interpreting the evidential weight of population assignments. We used Genogeographer for likelihood ratio (LR) calculations and Brazilians as examples of admixed individuals. Brazilians are a very heterogenous population representing a three-way admixture between Native Americans, Europeans, and Africans. Ancestry informative markers were typed in a total of 589 individuals from Brazil using the Precision ID Ancestry Panel. The Brazilians were assigned to six metapopulations (East Asia, Europe, Middle East, North Africa, South-Central Asia, Sub-Saharan Africa) defined in the Genogeographer software and LRs were calculated if the AIM profile was not an outlier in all metapopulations and simulated two-way (1:1) admixtures of the six metapopulations.Population assignments failed for 55% of the samples. These samples had significantly higher genetic contributions from East Asia, South-Central Asia and Sub-Saharan Africa, and significantly lower genetic contributions from Europe. Most of the individuals with population assignments were assigned to the metapopulations of Middle East (58%) or North Africa (36%), followed by Europe (4%), South-Central Asia (1%), and Sub-Saharan Africa (1%). For 8% of the samples, population assignments were only possible when assignments to simulated two-way (1:1) admixtures of the six metapopulations were considered. Most of these individuals were assigned to two-way admixtures of North Africa, South-Central Asia, or Sub-Saharan Africa.Relatively low median likelihood ratios (LRs<1000) were observed when comparing population likelihoods for Europe, Middle East, North Africa, South-Central Asia, or simulated 1:1 admixtures of these metapopulations. Comparisons including East Asian or Sub-Saharan African populations resulted in larger median LRs (LR>1010).The results suggested that the Precision ID Ancestry Panel provided too little information and that additional markers specifically selected for sub-continental differentiation may be required for accurate population assignment of admixed individuals. Furthermore, a Genogeographer database with additional populations including admixed populations would be advantageous for interpretation of admixed AIM profiles. It would likely increase the number of population assignments and illustrate alternatives to the most likely population, which would be valuable information for the case officer when writing the case report.

Population analysis of complete mitogenomes for 334 samples from El Salvador

The use of mitochondrial DNA (mtDNA) in the field of forensic genetics is widely spread mainly due to its advantages when identifying highly degraded samples. In this sense, massive parallel sequencing has made the analysis of the whole mitogenome more accessible, noticeably increasing the informativeness of mtDNA haplotypes. The civil war (1980–1992) in El Salvador caused many deaths and disappearances (including children) all across the country and the economic and social instability after the war forced many people to emigration. For this reason, different organizations have collected DNA samples from relatives with the aim of identifying missing people. Thus, we present a dataset containing 334 complete mitogenomes from the Salvadoran general population. To the best of our knowledge, this is the first publication of a nationwide forensic-quality complete mitogenome database of any Latin American country. We found 293 different haplotypes, with a random match probability of 0.0041 and 26.6 mean pairwise differences, which is similar to other Latin American populations, and which represent a marked improvement from the values obtained with just control region sequences. These haplotypes belong to 54 different haplogroups, being 91% of them of Native American origin. Over a third (35.9%) of the individuals carried at least a heteroplasmic site (excluding length heteroplasmies). Ultimately, the present database aims to represent mtDNA haplotype diversity in the general Salvadoran populations as a basis for the identification of people that disappeared during or after the civil war.

Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR

BackgroundInsertion and deletion (InDel) polymorphisms have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. MethodA rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. ResultsIt showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. ConclusionThis method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.

DNA Methylation: Bridging life’s experience and genetics within forensic scenario

DNA serving as the “blueprints of life” is a complex found in all biological cells carrying the genetic instructions for development and functioning of the organism. Inheritance of unique combination of genetic polymorphism results in DNA profile. In past few decades, the field of forensic genetics has witnessed several advancements from the radio-labelled DNA probes to the Short Tandem Repeats (STR) and Single Nucleotide Polymorphism (SNP) that have fostered the emergence of biological materials as most reliable evidences at a crime scene for the purpose of human identification but then, are inadequate in distinguishing monozygotic twins. Also, these techniques are of unavailing in providing informations such as tissue-specificity of the DNA source, phenotypic identification, and age estimation. Epigenetics, particularly DNA methylation is evolving as a promising technique to overcome such drawbacks of conventional profiling techniques.

Evaluation of microhaplotype panels for complex kinship analysis using massively parallel sequencing

In recent years, microhaplotypes (MHs) have become a research hotspot within the field of forensic genetics. Traditional MHs contain only SNPs that are closely linked within short fragments. Herein, we broaden the concept of general MHs to include short InDels. Complex kinship identification plays an important role in disaster victim identification and criminal investigations. For distant relatives (e.g., 3rd-degree), many genetic markers are required to enhance power of kinship testing. We performed genome-wide screening for new MH markers composed of two or more variants (InDel or SNP) within 220 bp based on the Chinese Southern Han from the 1000 Genomes Project. An NGS-based 67plex MH panel (Panel B) was successfully developed, and 124 unrelated individual samples were sequenced to obtain population genetic data, including alleles and allele frequencies. Of the 67 genetic markers, 65 MHs were, as far as we know, newly discovered, and 32 MHs had effective number of allele (Ae) values greater than 5.0. The average Ae and heterozygosity of the panel were 5.34 and 0.7352, respectively. Next, 53 MHs from a previous study were collected as Panel A (average Ae of 7.43), and Panel C with 87 MHs (average Ae of 7.02) was formed by combining Panels A and B. We investigated the utility of these three panels in kinship analysis (parent-child, full siblings, 2nd-degree, 3rd-degree, 4th-degree, and 5th-degree relatives), with Panel C exhibiting better performance than the two other panels. Panel C was able to separate parent-child, full-sibling, and 2nd-degree relative duos from unrelated controls in real pedigree data, with a small false testing level (FTL) of 0.11% in simulated 2nd-degree duos. For more distant relationships, the FTL was much higher: 8.99% for 3rd-degree, 35.46% for 4th-degree, and 61.55% for 5th-degree. When a carefully chosen extra relative was known, this may enhance the testing power for distant kinship analysis. Two twins from the Q family (2–5 and 2–7) and W family (3–18 and 3–19) shared the same genotypes in all tested MHs, which led to the incorrect conclusion that an uncle-nephew duo was classified as a parent-child duo. In addition, Panel C showed great capacity for excluding close relatives (2nd-degree and 3rd-degree relatives) during paternity tests. Among 18,246 real and 10,000 simulated unrelated pairs, none were misinterpreted as a relative within 2nd-degree at a log10(LR) cutoff of 4. The panels presented herein could provide supplementary power for the analysis of complex kinship.

Forty years of research and development on forensic genetics: A bibliometric analysis

The current study aims to investigate the research publication trends in the field of forensic genetics using Bibliometric analysis. An extensive search of the Scopus database was conducted to identify scholarly articles on forensic genetics published between 1977 and 2022, and a data set comprising 2945 articles was obtained. The analysis was carried out using VOSviewer, RStudio, MS Excel and MS Access to investigate the annual publication trend, most productive journals, organizations/authors/countries, authorship and citation patterns, most cited documents/articles and co-occurrence of keywords. The results revealed the first article in the field of forensic genetics was published in 1977. By the end of 1999, only 15 articles were published. Since then, there has been a considerable increase in the yearly number of publications and post-2006, there were more than 100 yearly published articles. USA, China, Spain, Germany and United Kingdom were found to be the most productive countries. Among various organizations, the Institute of Legal Medicine, Innsbruck Medical University, Austria was found to be the most productive organization. In terms of the number of publications and citations, Morling N. was found to be the most prolific author. The highest number of articles were published in Forensic Science International: Genetics, contributing about 34% of the total articles published in different sources/journals. The document with the highest number of citations was “HOMER N, 2008, PLOS GENET”, with a total of 750 citations. The most frequent keywords were forensic genetics and forensic science, followed by STR, population genetics, DNA, mt-DNA and DNA-typing. The results also revealed that there had been collaborative research among countries, organizations and authors, which helps in the exchange of ideas across disciplines, developing new skills, getting access to financial resources and generating quality results.

The GeFI (Italian Forensic Geneticists) code of conduct

The Ge.F.I. (Italian Forensic Geneticists) board has set up a working group aimed to develop the “Code of conduct of a Forensic Geneticist”. This group includes all practitioners working in the field of forensic genetics. The rules of the code of conduct apply to forensic geneticists belonging to the GeFI group and all of those sharing the GeFI’s mission, vision and values applicable to their professional practice, reciprocal relationship and relationship with third parties.

Applicability of the IrisPlex system for eye color prediction in an admixed population from Argentina.

Eye color prediction based on an individual's genetic information is of interest in the field of forensic genetics. In recent years, researchers have studied different genes and markers associated with this externally visible characteristic and have developed methods for its prediction. The IrisPlex represents a validated tool for homogeneous populations, though its applicability in populations of mixed ancestry is limited, mainly regarding the prediction of intermediate eye colors. With the aim of validating the applicability of this system in an admixed population from Argentina (n=302), we analyzed the six single nucleotide variants used in that multiplex for eye color and four additional SNPs, and evaluated its prediction ability. We also performed a genotype-phenotype association analysis. This system proved to be useful when dealing with the extreme ends of the eye color spectrum (blue and brown) but presented difficulties in determining the intermediate phenotypes (green), which were found in a large proportion of our population. We concluded that these genetic tools should be used with caution in admixed populations and that more studies are required in order to improve the prediction of intermediate phenotypes.

Typing of semen-containing mixtures using ARMS-based semen-specific CpG-InDel/STR markers.

Mixed traces are common biological materials found at crime scenes, and their identification remains a significant challenge in the field of forensic genetics. In recent years, DNA methylation has been considered as a promising approach for body fluid identification, and length polymorphic loci are still the preferred markers for personal identification. In this study, we used tissue-specific CpG sites with linked insertion or deletion (InDel) or short tandem repeat (STR) markers (CpG-InDel/STR) for both body fluid and individual identification. The tissue-specific CpG loci, which were all selected from the previous reports, were analyzed using a combination of bisulfite conversion and amplification refractory mutation system-multiprimer-PCR technology. InDels or STRs, which were selected within 400bp upstream or downstream of the semen-specific CpG loci, were analyzed using a capillary electrophoresis platform. Eventually, we successfully constructed a panel containing 17 semen-specific CpG-InDel/STR compound markers compassing 21 InDels/STRs, 3 body-fluid positive controls (vaginal secretion-, saliva-, and blood-specific CpG), and 1 gender identification locus. Using this panel, full genotyping of individuals could be obtained successfully with 50ng DNA input. Semen stains stored at room temperature for 7months and degraded samples that were heat treated for up to 6h were still identified efficiently. For semen containing mixed stains, it is also useful when the semen content is as low as 3.03%. Moreover, the cumulative discrimination power of this panel is 0.9999998. In conclusion, it is a robust panel enabling the validation of both the tissue source and individual identification of semen containing mixed stains and can be employed as an alternative solution for forensic case investigation.

Sequence Variations of 31 Υ-Chromosomal Short Tandem Repeats Analyzed by Massively Parallel Sequencing in Three U.S. Population Groups and Korean Population.

BackgroundRapidly mutating (RM) Y-chromosomal short tandem repeats (Y-STRs) have been demonstrated to increase the possibility of distinguishing between male relatives due to a higher mutation rate than conventional Y-STRs. Massively parallel sequencing (MPS) can be useful for forensic DNA typing as it allows the detection of sequence variants of many forensic markers. Here, we present sequence variations of 31 Y-STRs including nine RM Y-STRs (DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS570, DYS576, DYS612, and DYS627), their frequencies, distribution, and the gain in the number of alleles using MPS.MethodsWe constructed a multiplex MPS assay capable of simultaneously amplifying 32 Y-chromosomal markers, producing amplicons ranging from 85–274 bp. Barcoded libraries from 220 unrelated males from four populations—African Americans, Caucasians, Hispanics, and Koreans—were generated via two-step polymerase chain reaction and sequenced on a MiSeq system. Genotype concordance between the capillary electrophoresis (CE) and MPS method and sequence variation of Y-STRs were investigated.ResultsIn total, 195 alleles were increased by MPS compared to CE-based alleles (261 to 456). The DYS518 marker showed the largest increase due to repeat region variation (a 3.69-fold increase). The highest increase in the number of alleles due to single nucleotide polymorphisms in the flanking region was found in DYF399S1. RM Y-STRs had more diverse sequences than conventional Y-STRs. Furthermore, null alleles were observed in DYS576 due to primer-binding site mutation, and allele drop-outs in DYS449 resulted from low marker coverage of less than the threshold.ConclusionThe results suggest that the expanded and discriminative MPS assay could provide more genetic information for Y-STRs, especially for RM Y-STRs, and could advance male individualization. Compiling sequence-based Y-STR data for worldwide populations would facilitate the application of MPS in the field of forensic genetics and could be applicable in solving male-related forensic cases.

Research progress on application of microhaplotype in forensic genetics

As a novel genetic marker, microhaplotype can be applied in the field of forensic genetics. Microhaplotype has the advantages of high polymorphism, low mutation rate, no stutter products and short amplification fragments. Microhaplotype can effectively detect mixture, and quantitatively analyze the contributors of mixture. DNA with severe fragmentation can be successfully genotyped by microhaplotype. It can be used as ancestry informative marker to effectively divide the global continental population according to genetic structure. Microhaplotype system can provide more information than traditional short tandem repeat and help to identify complex relationships. It can provide new ideas for tumor source identification, cell line identification and prenatal paternity testing. Here we review the applications of microhaplotype, intending to provide references for forensic practice.

Developments in forensic DNA analysis.

The analysis of DNA from biological evidence recovered in the course of criminal investigations can provide very powerful evidence when a recovered profile matches one found on a DNA database or generated from a suspect. However, when no profile match is found, when the amount of DNA in a sample is too low, or the DNA too degraded to be analysed, traditional STR profiling may be of limited value. The rapidly expanding field of forensic genetics has introduced various novel methodologies that enable the analysis of challenging forensic samples, and that can generate intelligence about the donor of a biological sample. This article reviews some of the most important recent advances in the field, including the application of massively parallel sequencing to the analysis of STRs and other marker types, advancements in DNA mixture interpretation, particularly the use of probabilistic genotyping methods, the profiling of different RNA types for the identification of body fluids, the interrogation of SNP markers for predicting forensically relevant phenotypes, epigenetics and the analysis of DNA methylation to determine tissue type and estimate age, and the emerging field of forensic genetic genealogy. A key challenge will be for researchers to consider carefully how these innovations can be implemented into forensic practice to ensure their potential benefits are maximised.

Genetic analysis of sudden unexpected death cases: Evaluation of library preparation methods to handle heterogeneous sample material

Over the past years, next-generation sequencing (NGS) technologies revolutionized the possibilities in a broad range of application areas. Also in the field of forensic genetics, NGS continuously gained in importance and attentiveness. A significant number of sudden cardiac deaths (SCD) in the young is due to heritable arrhythmia syndromes emphasizing the need of examining the genetic basis in these cases also with regard to the identification of relatives and/or patients being at risk. As a result, high-throughput methods became of increasing value in molecular autopsy investigations enabling the analysis of a broad spectrum of genes. Most standard protocols are optimized for high-quality samples and frequently not directly applicable to challenging forensic sample material. In the present study, we intended to examine a comprehensive gene panel associated with SCD and inherited arrhythmogenic disorders. We compared three different hybridization-based library preparation technologies in order to implement a suitable NGS workflow for heterogeneous, forensic as well as diagnostic sample material. The results obtained indicated, that the Illumina technologies Nextera DNA Flex and TruSeq were compatible with samples exhibiting varying levels of degradation. In comparison, the TruSight method also resulted in good sequencing data, but seemed to be more dependent on DNA integrity. The preparation protocols evaluated in our study are not restricted to molecular autopsy investigations and might be helpful for and transferrable to further forensic research applications.

Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation.

The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.