Interpreting Mixture Profiles: Comparison between Precision ID GlobalFiler™ NGS STR Panel v2 and Traditional Methods.

Forensic investigation for the identification of offenders, recognition of human remains, and verification of family relationships requires the analysis of particular types of highly informative DNA markers, which have high discriminatory power and are efficient for typing degraded samples. These markers, called STRs (Short Tandem Repeats), can be amplified by multiplex-PCR (Polymerase Chain Reaction) allowing attainment of a unique profile through which it is possible to distinguish one individual from another with a high statistical significance. The rapid and progressive evolution of analytical techniques and the advent of Next-Generation Sequencing (NGS) have completely revolutionized the DNA sequencing approach. This technology, widely used today in the diagnostic field, has the advantage of being able to process several samples in parallel, producing a huge volume of data in a short time. At this time, although default parameters of interpretation software are available, there is no general agreement on the interpretation rules of forensic data produced via NGS technology. Here we report a pilot study aimed for a comparison between NGS (Precision ID GlobalFiler™ NGS STR Panel v2, Thermo Fisher Scientific, Waltham, MA, USA) and traditional methods in their ability to identify major and minor contributors in DNA mixtures from saliva and urine samples. A quantity of six mixed samples were prepared for both saliva and urine samples from donors. A total of 12 mixtures were obtained in the ratios of 1:2; 1:4; 1:6; 1:8; 1:10; and 1:20 between minor and major contributors. Although the number of analyzed mixtures is limited, our results confirm that NGS technology offers a huge range of additional information on samples, but cannot ensure a higher sensitivity in respect to traditional methods. Finally, the Precision ID GlobalFiler™ NGS STR Panel v2 is a powerful method for kinship analyses and typing reference samples, but its use in biological evidence should be carefully considered on the basis of the characteristics of the evidence.

Age Estimation with DNA: From Forensic DNA Fingerprinting to Forensic (Epi)Genomics: A Mini-Review.

Forensic genetics developed from protein-based techniques a quarter of a century ago and became famous as "DNA fingerprinting," this being based on restriction fragment length polymorphisms (RFLPs) of high-molecular-weight DNA. The amplification of much smaller short tandem repeat (STR) sequences using the polymerase chain reaction soon replaced RFLP analysis and advanced to become the gold standard in genetic identification. Meanwhile, STR multiplexes have been developed and made commercially available which simultaneously amplify up to 30 STR loci from as little as 15 cells or fewer. The enormous information content that comes with the large variety of observed STR genotypes allows for genetic individualisation (with the exception of identical twins). Carefully selected core STR loci form the basis of intelligence-led DNA databases that provide investigative leads by linking unsolved crime scenes and criminals through their matched STR profiles. Nevertheless, the success of modern DNA fingerprinting depends on the availability of reference material from suspects. In order to provide new investigative leads in cases where such reference samples are absent, forensic scientists started to explore the prediction of phenotypic traits from the DNA of the evidentiary sample. This paradigm change now uses DNA and epigenetic markers to forecast characteristics that are useful to triage further investigative work. So far, the best investigated externally visible characteristics are eye, hair and skin colour, as well as geographic ancestry and age. Information on the chronological age of a stain donor (or any sample donor) is elemental for forensic investigations in a number of aspects and has, therefore, been explored by researchers in some detail. Among different methodological approaches tested to date, the methylation-sensitive analysis of carefully selected DNA markers (CpG sites) has brought the most promising results by providing prediction accuracies of ±3-4 years, which can be comparable to, or even surpass those from, eyewitness reports. This mini-review puts recent developments in age estimation via (epi)genetic methods in the context of the requirements and goals of forensic genetics and highlights paths to follow in the future of forensic genomics.

Back to the Future - Part 2. Post-mortem assessment and evolutionary role of the bio-medicolegal sciences.

Part 2 of the review "Back to the Future" is dedicated to the evolutionary role of the bio-medicolegal sciences, reporting the historical profiles, the state of the art, and prospects for future development of the main related techniques and methods of the ancillary disciplines that have risen to the role of "autonomous" sciences, namely, Genetics and Genomics, Toxicology, Radiology, and Imaging, involved in historic synergy in the "post-mortem assessment," together with the mother discipline Legal Medicine, by way of its primary fundament, universally denominated as Forensic Pathology. The evolution of the scientific research and the increased accuracy of the various disciplines will be oriented towards the elaboration of an "algorithm," able to weigh the value of "evidence" placed at the disposal of the "justice system" as real truth and proof.

DNA Phenotyping: Snapshot of a Criminal.

Recent progresses in genomic technologies provide both opportunities and challenges to forensics.

Molecular DNA Analysis in Forensic Identification.

BACKGROUND: Serological and biochemical identification methods used in forensics have several major disadvantages, such as: long time in processing biological sample and lack of sensitivity and specificity. In the last 30 years, DNA molecular analysis has become an important tool in forensic investigations. DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples. METHODS: Forensic genetics, can provide information on the events which occurred at the crime scene or to supplement other methods of forensic identification. Currently, the methods used in identification are based on polymerase chain reaction (PCR) analyses. This method analyses the autosomal STRs, the Y-chromosome, and the mitochondrial DNA. RESULTS: Correlation of biological samples present at the crime scene with identification, selection, and the probative value factor is therefore the first aspect to be taken into consideration in the forensic genetic analysis. CONCLUSIONS: In the last decade, because of the advances in the field of molecular biology, new biomarkers such as: microRNAs (miR), messenger RNA (mRNA), and DNA methylation have been studied and proposed to be used in the forensic identifications of body fluids.

STR allele sequence variation: Current knowledge and future issues.

This article reviews what is currently known about short tandem repeat (STR) allelic sequence variation in and around the twenty-four loci most commonly used throughout the world to perform forensic DNA investigations. These STR loci include D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, SE33, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045. All known reported variant alleles are compiled along with genomic information available from GenBank, dbSNP, and the 1000 Genomes Project. Supplementary files are included which provide annotated reference sequences for each STR locus, characterize genomic variation around the STR repeat region, and compare alleles present in currently available STR kit allelic ladders. Looking to the future, STR allele nomenclature options are discussed as they relate to next generation sequencing efforts underway.

Genotyping and interpretation of STR-DNA: Low-template, mixtures and database matches-Twenty years of research and development.

The introduction of Short Tandem Repeat (STR) DNA was a revolution within a revolution that transformed forensic DNA profiling into a tool that could be used, for the first time, to create National DNA databases. This transformation would not have been possible without the concurrent development of fluorescent automated sequencers, combined with the ability to multiplex several loci together. Use of the polymerase chain reaction (PCR) increased the sensitivity of the method to enable the analysis of a handful of cells. The first multiplexes were simple: 'the quad', introduced by the defunct UK Forensic Science Service (FSS) in 1994, rapidly followed by a more discriminating 'six-plex' (Second Generation Multiplex) in 1995 that was used to create the world's first national DNA database. The success of the database rapidly outgrew the functionality of the original system - by the year 2000 a new multiplex of ten-loci was introduced to reduce the chance of adventitious matches. The technology was adopted world-wide, albeit with different loci. The political requirement to introduce pan-European databases encouraged standardisation - the development of European Standard Set (ESS) of markers comprising twelve-loci is the latest iteration. Although development has been impressive, the methods used to interpret evidence have lagged behind. For example, the theory to interpret complex DNA profiles (low-level mixtures), had been developed fifteen years ago, but only in the past year or so, are the concepts starting to be widely adopted. A plethora of different models (some commercial and others non-commercial) have appeared. This has led to a confusing 'debate' about the 'best' to use. The different models available are described along with their advantages and disadvantages. A section discusses the development of national DNA databases, along with details of an associated controversy to estimate the strength of evidence of matches. Current methodology is limited to searches of complete profiles - another example where the interpretation of matches has not kept pace with development of theory. STRs have also transformed the area of Disaster Victim Identification (DVI) which frequently requires kinship analysis. However, genotyping efficiency is complicated by complex, degraded DNA profiles. Finally, there is now a detailed understanding of the causes of stochastic effects that cause DNA profiles to exhibit the phenomena of drop-out and drop-in, along with artefacts such as stutters. The phenomena discussed include: heterozygote balance; stutter; degradation; the effect of decreasing quantities of DNA; the dilution effect.

Global spatiotemporal and genetic footprint of the H5N1 avian influenza virus.

BACKGROUND: Since 2005, the Qinghai-like lineage of the highly pathogenic avian influenza A virus H5N1 has rapidly spread westward to Europe, the Middle East and Africa, reaching a dominant level at a global scale in 2006. METHODS: Based on a combination of genetic sequence data and H5N1 outbreak information from 2005 to 2011, we use an interdisciplinary approach to improve our understanding of the transmission pattern of this particular clade 2.2, and present cartography of global spatiotemporal transmission footprints with genetic characteristics. RESULTS: Four major viral transmission routes were derived with three sources- Russia, Mongolia, and the Middle East (Kuwait and Saudi Arabia)-in the three consecutive years 2005, 2006 and 2007. With spatiotemporal transmission along each route, genetic distances to isolate A/goose/Guangdong/1996 are becoming significantly larger, leading to a more challenging situation in certain regions like Korea, India, France, Germany, Nigeria and Sudan. Europe and India have had at least two incursions along multiple routes, causing a mixed virus situation. In addition, spatiotemporal distribution along the routes showed that 2007/2008 was a temporal separation point for the infection of different host species; specifically, wild birds were the main host in 2005-2007/2008 and poultry was responsible for the genetic mutation in 2009-2011. "Global-to-local" and "high-to-low latitude" transmission footprints have been observed. CONCLUSIONS: Our results suggest that both wild birds and poultry play important roles in the transmission of the H5N1 virus clade, but with different spatial, temporal, and genetic dominance. These characteristics necessitate that special attention be paid to countries along the transmission routes.

DNA evidence: current perspective and future challenges in India.

Since the discovery of DNA fingerprinting technology in 1985 it has been used extensively as evidence in the court of law world-wide to establish the individual identity both in civil and criminal matters. In India, the first case of parentage dispute solved by the use of DNA fingerprinting technology was in 1989. Since then till date, the DNA technology has been used not only to resolve the cases of paternity and maternity disputes, but also for the establishment of individual identity in various criminal cases and for wildlife forensic identification. Since last half a decade, India is exercising to enact legislation on the use of DNA in the judicial realm and the draft 'Human DNA Bill-2012' is pending in the parliament. Largely, the promoters of forensic DNA testing have anticipated that DNA tests are nearly infallible and DNA technology could be the greatest single advance step in search for truth, conviction of the perpetrator, and acquittal of the innocent. The current article provides a comprehensive review on the status of DNA testing in India and elucidates the consequences of the admissibility of DNA as 'evidence' in the judicial dominion. In this backdrop of civil and criminal laws and changing ethical and societal attitudes, it is concluded that the DNA legislation in India and world-wide needs to be designed with utmost care.

Reflexiones criticas acerca de la ley de Identificación Genética Brasileña / No disponible

La ley 12.654, de 28 de mayo de 2012, implementó en Brasil la base de datos de los perfiles genéticos con finalidades criminales, con el objetivo de permitir la identificación genética de las personas investigadas y condenadas por «crímenes hediondos» o delitos dolosos practicados con violencia de naturaleza grave contra las personas. El presente trabajo tiene por objeto realizar un análisis crítico de las implicaciones procesales penales del funcionamiento de las bases de datos genéticos en el ordenamiento jurídico brasileño, así como las principales dificultades encontradas para la implementación de este mecanismo técnico-investigativo en nuestro país. Tales reflexiones son hechas a partir de la concepción de un modelo constitucional de proceso que tiene por finalidad la protección de derechos fundamentales. De esta forma, se analiza primeramente la noción de identidad en el Estado Democrático de Derecho, trazándose un panorama sobre la identificación criminal, como medida de intervención corporal, y como la jurisprudencia brasileña viene tratando las injerencias en el cuerpo humano. Finalmente, se analiza el régimen jurídico de los datos genéticos y el empleo de los bancos de datos de AND (AU)

The act 12.654 of May 28, 2012 implemented the database of genetic profiles for criminal purposes, in order to allow for the identification of individuals investigated and convicted of heinous crimes or intentional violent offenses of serious nature against other persons. The present work seeks to make a critical analysis of criminal procedural implications of the genetic databases in the Brazilian legal system and the main difficulties found in the implementation o such technical investigate mechanism in our country. These reflections are made from the idea of a constitutional process model that is intended to protect fundamental rights. Thus, it first analyzes the notion of identity in the democratic state of law, mapping out a perspective of criminal identification as a measure of body intervention as well as how Brazilian jurisprudence has been treating various interferences in the human body. Finally it discusses the legal regime of genetic data and the use of DNA databases (AU)

[The evaluation of the prospects for the application of mass-spectrometric analysis of the amplified DNA fragments for the purpose of forensic medical expertise].

The objective of the present study was to evaluate the prospects for the application of the mass-spectrometric analysis for the solution of the problems facing modern forensic-medical genetics as illustrated by the example of the new experimental multiplex approach to the typing of human DNA with the use of the complex PLEX-ID platform. The validation study involved all stages of the processing chain. The results of the study were used to develop the recommendations for the optimization of the analytical system being used. The comparative analysis of the experimental PLEX-ID technology and the traditional electrophoretic system for the analysis of polymorphism of amplified DNA fragments has demonstrated the potential advantages of the mass-spectrometric technique, such as the enhanced informative value of the forensic expert evaluation of polymorphism of STR-loci due to the possibility of identifying SNP and extension in them and therefore their allelic spectrum.

La identificación de personas mediante pruebas genéticas y bancos de perfiles de ADN / The identification of individuals through genetic testing and DNA profiling banks

No disponible

Derechos fundamentales afectados en la toma de muestras biológicas para la obtención de medios de pruebas de ADN válidos y eficaces, desde el punto de vista del Derecho interno y del Derecho de la Unión Europea / Fundamental rights affected by biological sampling to obtain valid and efficient DNA evidence from the point of view of national law and European Union law

No disponible

¿Es infalible la prueba pericial de ADN?: planteamiento de la cuestión / Is DNA expert evidence infalible? Approach to the question

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Contribuciones de la Comisión Nacional para el uso forense del ADN desde una perspectiva bioética / Contributions of the National Commission for the Forensic Use of DNA from a bioethical perspective

No disponible