Andrea Berti

Molecular identification of vaginal fluid by microbial signature.

The discrimination of body fluids in forensic examinations can play an important role in crime scene reconstruction. Conventional methods rely on the detection of antigens or enzymatic activity, limiting detection sensitivity and specificity, particularly on old forensic samples. Methods based on human RNA analysis are not easily applicable to samples exposed to harsh and degrading environments. An alternative approach based on the identification of prokaryotic genomes was developed. Specific bacterial communities are characteristic typical of different human non-sterile body fluids: the molecular characterization of a microbial signature, and not the typing of single bacterial species, can effectively lead to univocal identification of these fluids. A multiplex real time PCR assay was developed using oligonucleotide mixtures targeting genomes specific for a selected group of bacteria. Microflora DNA (mfDNA) was extracted from vaginal, oral and fecal clinical swabs. In addition forensic samples were processed. Vaginal samples showed a strong specific signal for bacteria of the female genital tract. Oral samples clearly showed signal for bacteria present in saliva, and in fecal samples the main signal was from Enterococcaceae. Vaginal casework samples showed results comparable to freshly collected ones; moreover the DNA extracted was successfully used for STR typing. Also mixtures of body fluids were analyzed, providing a microbiological signature compatible with the presence of microbes of oral, fecal and vaginal origin. The presented method can be useful in identifying biological fluids, and it is based on DNA technologies already available in forensic laboratories and feasible for further high throughput automation.

Allele frequencies of the new European Standard Set (ESS) loci in the Italian population

Allele frequencies of five new STR loci (D22S1045, D10S1248, D1S1656, D12S391, D2S441) included in the new European Standard Set (ESS) were calculated in a sample of 209 unrelated Italians with the Powerplex ESI 17 system (Promega Corporation, Madison, WI). Forensic and population indices were estimated. Samples were collected from unrelated individuals in 19 different Italian regions following informed consent. DNA was extracted from saliva by Chelex method [1]. A prototype version of the PowerPlex ESI 17 (Promega Corporation, Madison, WI) was used to amplify individuals’ DNA according to manufacturer’s recommendations. This multiplex contains 17 loci of which five are novel STR loci (D22S1045, D10S1248, D1S1656, D12S391, D2S441) included in the new European Standard Set (ESS) [2,3]. The other loci include the amelogenin, D3S1358, D19S433, D2S1338, D16S539, D18S51, TH01, vWA, D21S11, D8S1179, FGA and SE33. PCR products were analysed by capillary electrophoresis in an ABI 3130xl Genetic analyzer (Applied Biosystem, Foster City, CA). Allele assignment was carried out by comparison with reference sequenced ladders (Promega Corporation, Madison, WI). Arlequin software ver 3.0 [4] was used to calculate allele frequencies, population pairwise genetic distances (FST), expected heterozigosity (He), observed heterozigosity (Ho), and also to assess departures from Hardy–Weinberg equilibrium. Statistical parameters of forensic interest (Power of Discrimination, Power of Exclusion and Matching Probability) were calculated using PowerStats v1.2 (Promega Corporation, USA) software package [5]. The laboratory participates in the quality control initiatives of the GEDNAP (German DNA Profiling) group [6] and [7] (http:// www.gednap.org). The whole genotype data set, allele frequencies and forensic indices are available as an e-component. Deviation from Hardy–Weinberg equilibrium has been detected only for D18S51, even after a Bonferroni correction. The combined power of exclusion (PE) and power of discrimination (PD) for the sixteen studied loci were 0.999999935 and 0.999999999, respectively. Based on heterozygosity and polymorphic information content (PIC), SE33 may be considered as the most informative loci. The exclusion of this locus slightly reduced the PE estimate (0.999999555). The PD value is similar for those calculated on a different Italian population set using the PowerPlex16 multiplex system (Promega Corporation, Madison, WI), the Identifiler kit (Applied Biosystems) and for the markers included in the US Combined DNA Index System (CODIS) [8–10], while for the PE, the value obtained with the PowerPlex ESI 17 System (Promega Corporation, Madison, WI) is higher. No pair of loci

The environmental biological signature: NGS profiling for forensic comparison of soils

The identification of the source of a specific soil sample is a crucial step in forensic investigations. Rapid advances in next generation sequencing (NGS) technology and the strong reduction of the cost of sequencing have recently opened new perspectives. In the present work a metabarcoding approach has been successfully applied to forensic and environmental soil samples, allowing the accurate and sensitive analysis of microflora (mfDNA), plants, metazoa, and protozoa DNA. The identification of the biological component by DNA metabarcoding is a strong element for the discrimination of samples geologically very similar but coming for distinct environments.

Forensic genetic value of a 27 Y-STR loci multiplex (Yfiler® Plus kit) in an Italian population sample

The analysis of Y chromosome short tandem repeat (Y-STR) haplotypes provides important information that can be used for investigative purposes and in population studies. The Yfiler(®) Plus PCR Amplification kit (Yfiler(®) Plus, Thermo Fisher Scientific, Waltham, MA, USA) allows the multiplex amplification of 27 Y-STRs, including 7 rapidly mutating markers (RM Y-STRs). In this study, 203 unrelated males from Italy, which were subdivided into 4 different geographical groups (North, Center, South and Sardinia) were analyzed. Several intra-population diversity indexes were computed and compared to those obtained using only loci either from the minimal haplotype or the 17-plex (Yfiler(®), Thermo Fisher Scientific, Waltham, MA, USA). In addition, inter-population diversity analysis (RST) among the four Italian samples was performed. The same analysis was also used to compare the Italian sub-sets to other European populations where the Yfiler(®) Plus haplotype frequency data were available. The Sardinians were significantly differentiated from the other three Italian groups, thus requiring a specific sub-national Y-STR haplotype database. The Yfiler(®) Plus kit showed a high power of discrimination which is useful for criminal investigations, principally due to the inclusion of RM Y-STRs.

Expression of Seminal Vesicle-Specific Antigen in Serum of Lung Tumor Patients

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

Forensic data and microvariant sequence characterization of 27 Y-STR loci analyzed in four Eastern African countries

By using the recently introduced 6-dye Yfiler® Plus multiplex, we analyzed 462 males belonging to 20 ethnic groups from four eastern African countries (Eritrea, Ethiopia, Djibouti and Kenya). Through a Y-STR sequence analysis, combined with 62 SNP-based haplogroup information, we were able to classify observed microvariant alleles at four Y-STR loci as either monophyletic (DYF387S1 and DYS458) or recurrent (DYS449 and DYS627). We found evidence of non-allelic gene conversion among paralogous STRs of the two-copy locus DYF387S1. Twenty-two diallelic and triallelic patterns observed at 13 different loci were found to be significantly over-represented (p<10-6) among profiles obtained from cell lines compared to those from blood and saliva. Most of the diallelic/triallelic patterns from cell lines involved recurrent mutations at rapidly mutating loci (RM Y-STRs) included in the multiplex (p<10-2). At haplotype level, intra-population diversity indices were found to be among the lowest so far reported for the Yfiler® Plus, while statistically significant differences among countries and ethnic groups were detected when considering haplotype frequencies alone (FST) or by using molecular distances among haplotypes (ΦST). The strong population subdivision observed is probably the consequence of the patrilineal social organization of most eastern African ethnic groups, and suggests caution in the use of country-based haplotype frequency distributions for forensic inferences in this region.

The SNPs in the human genetic blueprint era

The analysis of human genetic variability can lead to the comprehension of medical issues and to the development of personalized therapeutic protocols. Single nucleotide polymorphisms, are the most common type of human genetic variation and have been associated to disease development and phenotype forecasting. The recent technologies for DNA sequencing and bioinformatic analysis are now giving the opportunity to develop new diagnostic and prevention approaches also through health promotion protocols. The genetic data management is at the same time underlining technical limitations and old ethical issues.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles.

Pet fur or fake fur? A forensic approach

BackgroundIn forensic science there are many types of crime that involve animals. Therefore, the identification of the species has become an essential investigative tool. The exhibits obtained from such offences are very often a challenge for forensic experts. Indeed, most biological materials are traces, hair or tanned fur. With hair samples, a common forensic approach should proceed from morphological and structural microscopic examination to DNA analysis. However, the microscopy of hair requires a lot of experience and a suitable comparative database to be able to recognize with a high degree of accuracy that a sample comes from a particular species and then to determine whether it is a protected one. DNA analysis offers the best opportunity to answer the question, ‘What species is this?’ In our work, we analyzed different samples of fur coming from China used to make hats and collars. Initially, the samples were examined under a microscope, then the mitochondrial DNA was tested for species identification. For this purpose, the genetic markers used were the 12S and 16S ribosomal RNA, while the hypervariable segment I of the control region was analyzed afterwards, to determine whether samples belonged to the same individual.ResultsMicroscopic examination showed that the fibres were of animal origin, although it was difficult to determine with a high degree of confidence which species they belonged to and if they came from a protected species. Therefore, DNA analysis was essential to try to clarify the species of these fur samples.ConclusionsMacroscopic and microscopic analysis confirmed the hypothesis regarding the analyzed hair belonging to real animals, although it failed to prove with any kind of certainty which actual family it came from, therefore, the species remains unknown. Sequence data analysis and comparisons with the samples available in GenBank showed that the hair, in most cases, belonged to the Canidae family, and in one case only to Felidae.

Traces of forgotten historical events in mountain communities in Central Italy: A genetic insight

Analysis of human genetic variation in mountain communities can shed light on the peopling of mountainous regions, perhaps revealing whether the remote geographic location spared them from outside invasion and preserved their gene pool from admixture. In this study, we created a model to assess genetic traces of historical events by reconstructing the paternal and maternal genetic history of seven small mountain villages in inland valleys of Central Italy.