Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Evaluation of a microhaplotypes panel for forensic genetics using massive parallel sequencing technology

Massive parallel DNA sequencing (MPS) makes it possible to explore a new type of genetic marker, known as microhaplotypes or microhaps. These loci were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Microhaplotypes loci, based on 89 loci in ALFRED, were selected and their genetic variations in 100 Italian individuals were evaluated by using MPS, in order to make inference about utility of a set of microhaps in forensic genetics. After MPS, the panel was reduced to 87 microhaps, comprised of 266 different SNPs and spread across 22 human autosomes. Genotype and haplotype frequencies were estimated, as well as the effective number of alleles at each locus (Ae), which relates to the usefulness of the locus in resolution of relationships and deconvolution of DNA mixtures. Overall, the Ae values for the 87 microhaps range from 1.010 to 8.344, with about 80% showing values greater than 2.0. Noteworthy, 32 microhaps display Ae values greater than 3.0 and 18 loci Ae above 4.0. To explore the suitability of microhaplotypes in mixture deconvolution, the probability of detecting a mixture, as a function of Ae, was inferred for different groups of loci. Considering the fourteen loci with Ae between 3.0 and 3.999 the probability of detecting a mixture was at least 0.99973, while considering the ten loci with Ae between 4.0 and 4.999 the probability was at least 0.99998. Moreover, when considering just the six loci with Ae between 5.0 and 5.999 the probability of detecting a mixture was at least 0.99984, while when considering just the two loci with Ae above 6 the probability was 0.97228. Combining these 32 MH loci, the theoretical probability of detecting a mixture was 0.999999999999973. These results make the subset of 32 loci with Ae above three informative for mixture resolution. The individual matching probabilities (PI) of the 87 microhaps ranged from 0.032 to 0.9802. Considering the 32 microhap loci with Ae greater than 3.0, the cumulative PI value was 1.6 × 10-33, while considering the 18 microhap loci with Ae above 4.0, the cumulative PI value was 2.34 × 10-21. Overall the results of this study confirmed the utility of microhaps in forensic genetics

Spleen nodules: a potential hallmark of Visceral Leishmaniasis in young children

open8noBACKGROUND: Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum in the Mediterranean basin, and is associated with considerable morbidity and mortality. Infantile VL may begin suddenly, with high fever and vomiting, or insidiously, with irregular daily fever, anorexia, and marked splenomegaly. Delays in diagnosis of VL are common, highlighting the need for increased awareness of clinicians for VL in endemic European countries. CASE PRESENTATION: We report 4 cases of young children in northern Italy presenting with persistent fever of unknown origin and diagnosed with VL by serological and molecular methods. At the time of diagnosis, these patients showed an unusual echographic pattern characterized by multiple iso-hypoechoic nodules associated with splenomegaly. CONCLUSION: We suggest that detection of spleen nodules represents a signature of VL in infants, thus helping to diagnose systemic Leishmania infantum infection in children.openFraia Melchionda;Stefania Varani;Filomena Carfagnini;Tamara Belotti;Trentina Di Muccio;Roberto Tigani;Rosalba Bergamaschi;Andrea PessionFraia Melchionda;Stefania Varani;Filomena Carfagnini;Tamara Belotti;Trentina Di Muccio;Roberto Tigani;Rosalba Bergamaschi;Andrea Pessio

Exploring the usefulness of microhaplotypes in forensic identification using massive parallel sequencing technology

BACKGROUND: Microhaplotypes or microhaps (MH) were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, genotyping of degraded DNA, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. In order to make inference about a set of microhaps useful in forensic casework with low amount of degraded DNA and useful in kinship analysis, several microhaps were tested by massive parallel sequencing (MPS) assay. METHODS: We have investigated the effectiveness of 29 microhaps in a set of real forensic samples together with artificially degraded DNAs. Moreover, we explore the informativeness of 87 microhaplotypes in relationship analysis through a simulation of different kinship testing scenarios typically encountered in forensic identi\ufb01cation. RESULTS: The MPS coverage analysis showed a good performance of the designed panel. Full profiles could be obtained with 0.1 ng of input DNA even with highly degraded samples. The increment of the number of PCR cycles does not result in an improvement in genotyping results in samples with low amounts of DNA, as the increase of drop-in and drop-out events were observed at 25 number of PCR cycles. No correlation between amplicons size and occurrence of drop-outs and drop-ins was observed. Kinship simulations showed that full siblings and half siblings relationships would be readily distinguished respect unrelated condition using the 87 microhaps panel. CONCLUSIONS: Results shown that microhaps could be a powerful tool for individual identification, relationship resolution and that they are sensitive and reliable in degraded DNA typing

Validation of a universal DNA extraction method for human and microbiAL DNA analysis

The study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). Before a new technology is accepted by the forensic science, it requires an initial validation phase. The aim of our study was to evaluate if the DNA IQ™ Casework Pro Kit for Maxwell® 16 (Promega) is suitable for microbial DNA extraction, without modifications. Ten bacterial strains were selected and subjected to the GenElute Bacterial Genomic DNA extraction protocol (Sigma-Aldrich) and to the DNA IQ™ Casework Pro Kit for Maxwell® 16 protocol. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Reporter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the Promega kit, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited

Dealing with low amounts of degraded DNA: Evaluation of SNP typing of challenging forensic samples by using massive parallel sequencing

A set of eighty-two forensic samples with different levels of degradation, as well five in vitro damaged samples were analyzed by the Precision ID Identity Panel. PCR amplifications were performed with scalar amount of DNA (from 1 ng to 12 pg) and through different number of cycles. A minimum coverage of 50 x was adopted for \u201clocus call\u201d. Very informative profiles (based on about 65\u201370% of the loci) were obtained even in highly degraded samples when the amount of template range from 0.1 to 1.0 ng. When dealing with low amount of degraded DNAs, no more than half of the loci were typed, and the risk of mistyping (due to drop out phenomena) increased dramatically. The employment of a high number of PCR cycles is discussed

Total body skin examination for skin cancer screening in patients with focused symptoms

The value of total body skin examination (TBSE) for skin cancer screening is controversial