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From a criminalistic point of view, the accurate dating of biological traces found at the crime scene, together with its compatibility with the estimated crime perpetration timeframe, enables to limit the number of suspects by assessing their alibis and clarifying the sequence of events. The present study delineates, for the first time, the possibility of dating biological fluids such as semen and urine, as well as blood traces, by using a novel non-destructive analytical strategy based on hyperspectral imaging in the near infared region (HSI-NIR), coupled with multivariate regression methods. Investigated aspects of the present study include not only the progressive degradation of the biological trace itself, but also the effects of its interactions with the support on which it is absorbed, in particular the hydrophilic vs. hydrophobic character of fabric tissues. Results are critically discussed, highlighting potential and limitations of the proposed approach for a practical implementation.

Y chromosome short tandem repeats (Y-STRs) typing is becoming increasingly popular in forensic casework mainly because it allows the recovery of male-specific genetic information from severely unbalanced male-female DNA mixtures. The relatively low discrimination power of conventional Y-STR multiplexes, due to linkage disequilibrium among polymorphic loci, has been partially overcome by the introduction of rapidly mutating Y microsatellites (RM Y-STRs) with mutation rates exceeding 1 × 10

To exploit the possibility to use single cells to deconvolute mixed Forensic samples it’s necessary to extend Forensic STR kit optimization to input levels of 6 pg. In this study we tested and compared three different commercially available kits, each at different cycling conditions, to identify the best conditions to obtain the most informative profiles from single cells or very small pools of cells, isolated from casework samples.

BackgroundDNA metabarcoding and massive parallel sequencing are valuable molecular tools for the characterization of environmental samples. In forensic sciences, the analysis of the sample’s fungal population can be highly informative for the estimation of post-mortem interval, the ascertainment of deposition time, the identification of the cause of death, or the location of buried corpses. Unfortunately, metabarcoding data analysis often requires strong bioinformatic capabilities that are not widely available in forensic laboratories.ResultsThe present paper describes the adoption of a user-friendly cloud-based application for the identification of fungi in typical forensic samples. The samples have also been analyzed through the QIIME pipeline, obtaining a relevant data concordance on top genus classification results (88%).ConclusionsThe availability of a user-friendly application that can be run without command line activities will increase the popularity of metabarcoding fungal analysis in forensic samples.

Y chromosome short tandem repeats (Y-STRs) are commonly used to identify male lineages for investigative and judicial purposes and could represent the only source of male-specific genetic information from unbalanced female-male mixtures. The Yfiler Plus multiplex, which includes twenty conventional and seven rapidly-mutating Y-STRs, represents the most discriminating patrilineal system commercially available to date. Over the past five years, this multiplex has been used to analyze several Eurasian populations, with a reported discrimination capacity (DC) approaching or corresponding to the highest possible value. However, despite the inclusion of rapidly mutating Y-STRs, extensive haplotype sharing was still reported for some African populations due to a number of different factors affecting the effective population size. In the present study, we analyzed 27 Y-STRs included in the Yfiler Plus multiplex and 82 Y-SNPs in central Sahel (northern Cameroon and western Chad), an African region characterized by a strong ethnic fragmentation and linguistic diversity. We evaluated the effects of population sub-structuring on genetic diversity by stratifying a sample composed of 431 males according to their ethnicity (44 different ethnic groups) and urbanization degree (four villages and four towns). Overall, we observed a low discrimination capacity (DC = 0.90), with 71 subjects (16.5 %) sharing 27 Y-STR haplotypes. Haplotype sharing was essentially limited to subjects with the same binary haplogroup, coming from the same location and belonging to the same ethnic group. Haplotype sharing was much higher in rural areas (average DC = 0.83) than urban settlements (average DC = 0.96) with a significant correlation between DC and census size (r = 0.89; p = 0.003). Notably, we found that genetic differentiation between villages from the same country ( Φ ST = 0.14) largely exceeded that found among countries ( Φ ST = 0.02). These findings have important implications for the choice of the appropriate reference population database to evaluate the statistical relevance of forensic Y-haplotype matches.

The importance of detecting minute biological traces in forensic evidences feeds the continuous interest towards the development of new dedicated technologies both sensitive and reliable. The present study describes the opportunity to combine chemical properties derived from NIR signals with spatial features typical of RGB images by means of hyperspectral imaging (HSI). An analytical procedure based on HSI data collection and their multivariate processing followed by normalized difference images (NDI) is proposed as a screening method to highlight otherwise invisible traces of biological fluids on different supports in view of their collection for DNA analysis. The pattern features identified inside the NDI provided insight into the nature of the biological trace, on the basis of the wavelength at which the stain is highlighted and irrespective of the support on which the stain is deposited. In particular, the procedure allowed to detect and distinguish traces (i.e., 10 and 20 μL volumes) of dehydrated blood, urine, and semen on glass, paper, cotton, denim and polyblend fabric. Beside the simulated specimens used to develop and test the protocol, its robustness was demonstrated also on real and unknown validation samples, confirming its feasibility in some real case studies. An interesting evolution of the proposed strategy is to lay the scientific foundations for the development of a handheld device directly applicable in field.

Bloodstains are a widespread kind of biological evidence at the crime scene and one of the most used reagents for the presumptive identification of blood for forensic purposes is tetramethyl-benzidine. We have introduced and validated the tetramethylbenzidine-based Combur 3 Test® E (Roche Diagnostics Corporation, Basel, Switzerland), a colorimetric catalytic test based upon the detection of the peroxidase-like activity of the hemoglobin, due to its high sensitivity, easiness of use and capability to maintain the complete structural and morphological integrity of the bloodstain. Analytical performances related to a forensic use of the test and the suitable applicability to the presumptive detection of bloodstains when extremely diluted, aged, mixed with several substances and deposited over a plethora of substrates was reliably proved. In addition, possible positive interferences of the test chemicals on the subsequent Short Tandem Repeats (STRs) DNA typing analyses, especially in Low-Template DNA (LT DNA) conditions, was evaluated. While the Combur 3 Test® E showed the same chemical interference drawbacks as other presumptive tests for blood as for the low specificity, we demonstrated that its format and our suggested protocol of use make it appropriate for the forensic presumptive detection of blood, better performing and much easier to use than other analogous presumptive tests and usually compatible with the following STRs DNA typing analyses.

Searching a national DNA database with complex and incomplete profiles usually yields very large numbers of possible matches that can present many candidate suspects to be further investigated by the forensic scientist and/or police. Current practice in most forensic laboratories consists of ordering these 'hits' based on the number of matching alleles with the searched profile. Thus, candidate profiles that share the same number of matching alleles are not differentiated and due to the lack of other ranking criteria for the candidate list it may be difficult to discern a true match from the false positives or notice that all candidates are in fact false positives. SmartRank was developed to put forward only relevant candidates and rank them accordingly. The SmartRank software computes a likelihood ratio (LR) for the searched profile and each profile in the DNA database and ranks database entries above a defined LR threshold according to the calculated LR. In this study, we examined for mixed DNA profiles of variable complexity whether the true donors are retrieved, what the number of false positives above an LR threshold is and the ranking position of the true donors. Using 343 mixed DNA profiles over 750 SmartRank searches were performed. In addition, the performance of SmartRank and CODIS were compared regarding DNA database searches and SmartRank was found complementary to CODIS. We also describe the applicable domain of SmartRank and provide guidelines. The SmartRank software is open-source and freely available. Using the best practice guidelines, SmartRank enables obtaining investigative leads in criminal cases lacking a suspect.

Metabarcoding through Next Generation Sequencing (NGS) has revolutionized environmental biological studies. The availability of this technical approach has opened the opportunity for a systematic implementation of fungal metabarcoding analysis in forensics, where standardized, sensitive and reproducible protocols are highly desirable. In the present paper, a pipeline including a semi-automated molecular protocol and user-friendly bioinformatics tools are applied to several kinds of environmental samples and forensic caseworks. The identification of fungi that characterize specific environments (like Aspergillus for indoor walls, or Penicillium, Debaryomices and Wickerhamomyces for food storage) can be informative for the provenance of samples. In some situations, fungal analysis cannot allow the identification of a defined environment but seems useful to cluster samples with similar provenance. Based on these considerations, fungal analysis can be included in a wider process of non-human DNA identification in order to provide clues on sample provenance.

The capability to achieve biogeographic ancestry (BGA) information from DNA profiles have been largely explored in forensic genetics because of its potential usefulness in providing investigative clues. For law enforcement and security purposes, when genetic data have been obtained from unknown evidence, but no reference samples are available and no hints come out from DNA databases, it would be extremely useful at least to infer the ethno-geographic origin of the stain donor by just examining traditional STRs DNA profiles. Current protocols for ethnic origin estimation using STRs profiles are usually based on Principal Component Analysis approaches and Bayesian methods. The present study provides an alternative approach that involves the use of target multivariate data analysis strategies for estimation of the BGA information from unknown biological traces. A powerful multivariate technique such as Partial Least Squares-Discriminant Analysis (PLS-DA) has been applied on NIST U.S. population datasets containing, for instance, the allele frequencies of African-American, Asian, Caucasian and Hispanic individuals. PLS-DA approach provided robust classifications, yielding high sensitivity and specificity models capable of discriminating the populations on ethnic basis. Finally, a real casework has been examined by extending the developed model to smaller and more geographically-restricted populations involving, for instance, Albanian, Italian and Montenegrian individuals.

DNA typing and genetic profile data interpretation are among the most relevant topics in forensic science; among other applications, genetic profile's capability to distinguish biogeographic information about population groups, subgroups and affiliations have been largely explored in the last decade. In fact, for investigative and intelligence purposes, it is extremely useful to identify subjects and estimate their biogeographic origins by examining the recovered DNA profiles from evidence on a crime scene. Current approaches for BiogeoGraphic Ancestry (BGA) estimation using STRs profiles are usually based on Bayesian methods, which quantify the evidence in terms of likelihood ratio, supporting or not the hypothesis that a certain profile belongs to a specific ethnic group. The present study provides an alternative approach to the likelihood ratio method that involves multivariate data analysis strategies for the estimation of multiple populations. Starting from the well-known NIST US autosomal STRs dataset involving African-American, Asian, and Caucasian individuals, and moving towards further and more geographically restricted populations (such as Northern Africans vs sub-Saharan Africans, Afghans vs Iraqis and Italians vs Romanians), powerful multivariate techniques such as Sparse and Logistic Principal Component Analysis (SL-PCA), Sparse Partial Least Squares-Discriminant Analysis (sPLS-DA) and Support Vector Machines (SVM) were employed and their discriminating power was also compared. Both sPLS-DA and SVM techniques provided robust classifications, yielding high sensitivity and specificity models capable of discriminating populations on ethnic basis. This application may represent a powerful and dynamic tool for law enforcement agencies whenever a standard autosomal STR profile is obtained from the biological evidence collected at a crime scene or recovered during mass-disaster and missing person investigations.

Recently, thirteen rapidly mutating Y-STRs (RM Y-STRs), characterized by a mutation rate higher than 10−2/STR/generation, have been proven to be extremely useful in distinguishing among close male relatives. Six of these RM-YSTRs have been included in the Yfiler™ Plus PCR Amplification Kit, which shows the highest discriminatory power among Y-STR commercial multiplex currently available. In this study, we used Yfiler Plus to analyze 1437 males from eastern, northern and sub-Saharan Africa (462, 477 and 498 subjects, respectively). 242 out of 1437 subjects were found to share 102 Y-STR haplotypes, resulting in a low discrimination capacity (DC = 0.90) as compared to other continental regions. With few exceptions, Y-STR haplotype sharing was limited to subjects coming from the same country and ethnic group, and belonging to the same binary Y-SNP haplogroup, suggesting possible familial relationships along the male lineage. In order to evaluate the presence of hidden familial relationships, all the 242 subjects sharing a Y-STR haplotype were further genotyped for 16 autosomal STRs using the AmpFlSTR® NGM SElect™ PCR Amplification Kit. Blind search and simulation test analyses for kinship using the Familias software revealed the presence of a low proportion of close relatives (second degree or closer) in our sample set. These findings show that close relatedness explains only a relatively small proportion of the observed Y-STR haplotype sharing, suggesting that a higher number of RM Y-STRs should be included in commercial kits to improve the discrimination power of male-specific markers in regions characterized by high levels of endogamy.

The identification of vaginal fluids is crucial in forensic science. Several molecular protocols based on PCR amplification of mfDNA (microflora DNA) specific for vaginal bacteria are now available. Unfortunately mfDNA extraction and PCR reactions require manual optimization of several steps. The aim of present study was the verification of a partial automatization of vaginal fluids identification through two instruments widely diffused in forensic laboratories: EZ1 Advanced robot and Rotor Gene Q 5Plex HRM. Moreover, taking advantage of 5-plex thermocycler technology, the ForFluid kit performances were improved by expanding the mfDNA characterization panel with a new bacterial target for vaginal fluids and with an internal positive control (IPC) to monitor PCR inhibition. Results underlined the feasibility of a semi-automated extraction of mfDNA using a BioRobot and demonstrated the analytical improvements of the kit.

During the Second World War, on 24th March 1944, 335 Italians were massacred near Rome by the occupying forces of Nazi Germany. Four months later forensic examination led to the identification of 323 out of 335 victims. After approximately 60 years, the identification of the remaining unidentified twelve victims began with anthropological and genetic analysis carried out by a team of Italian forensic experts. Anthropological analysis was performed in field in order to confirm the sex of each victim and verify the presence of only one individual in each grave for a correct sampling. Selected bone fragments for each individual were then collected and transferred to the laboratory for genetic analysis. Although the anthropological ante mortem information was limited, morphological and metrical data was collected for a possible future identification of the victims. Subsequently, the typing of autosomal loci, Y-STR and mtDNA D-loop region of all bone and available reference samples was conducted. LR and cumulative LRs obtained from autosomal STR and Y-STR results confirmed the alleged relationship between three victims and their relatives with values over 104 (one sample) and 106 (two samples). Therefore, the genetic analysis offered the families the possibility of replacing the number of the grave with the name of the victim.

The forensic application of the luminol chemiluminescence reaction is reviewed. Luminol has been effectively employed for more than 40 years for the presumptive detection of bloodstains which are hidden from the naked eye at crime scenes and, for this reason, has been considered one of the most important and well-known assays in the field of forensic sciences. This review provides an historical overview of the forensic use of luminol, and the current understanding of the reaction mechanism with particular reference to the catalysis by blood. Operational use of the luminol reaction, including issues with interferences and the effect of the luminol reaction on subsequent serological and DNA testing is also discussed.

Allele frequencies for the 15 autosomal STR loci included in the AmpFlSTR((R)) IdentifilerTM PCR Amplification Kit panel from Applied Biosystems (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, CSF1PO, D19S433, D2S1338, D16S539) and several statistical parameters were estimated from a sample of 103 unrelated individuals, mostly Shia and Sunni Arabs, living in most of central and southern Iraq provinces. We compared the allele frequency spectrum detected in the Iraqi population to allele frequencies from 11 other data sets from published studies of individuals from Turkey, Iraqi-Kurdistan, Saudi Arabia, Arab Emarates, Oman, Iran, Syria, and Jordan. Significant global differences in allele frequencies were detected in 9 of the 11 comparisons following sequential Bonferroni corrections. Comparisons with the two independent panels from Saudi Arabia were not significant after applying Bonferroni corrections, however, low P-values (P0.05) associated with these two contrasts nonetheless suggested that at least slight genetic differences between populations may exist.

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.

A commercially available semen detection kit, Nanotrap Sg, which employs a one-step detection test based on immunochromatographic assay for the semenogelin protein, was evaluated for profiling male-specific DNA in sexual assault casework samples. While semen diluted with phosphate-buffered saline held and kept at 4 degrees C for 1 week showed a relatively strong signal intensity with Nanotrap Sg, the signal intensity was decreased by dilution after storage at 4 degrees C or freezing and thawing repeated more than three times. The reproducibility of Nanotrap Sg was tested on a total of 174 sexual assault casework samples from three forensic laboratories using intra- and interassay and no variation was observed in the semenogelin (Sg) signal. The positive signal ratio was 12.6% higher for prostate-specific antigen immunochromatographic membrane tests than Nanotrap Sg. Although spermatozoa were not confirmed in 61 (35%) out of 174 samples, Sg-positive signals could be detected from 41 (67%) of the 61 samples. Female genetic profiles could be observed in 95% of the samples, which tested negative for Sg on the Nanotrap Sg test, but no male genetic profiles could be observed. These results suggest that Nanotrap Sg can positively identify samples containing male DNA even in the absence of detectable intact spermatozoa. Further, Sg-positive signals identified samples for which male-specific DNA profiling could be performed, even if no sperm could be detected from the sample. The potential of Nanotrap Sg for identifying forensic samples with male-specific DNA was clearly demonstrated.

Background In 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. Results Microscopic 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. Conclusions Macroscopic 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.

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.

Chemical maceration is a useful procedure to remove soft tissues and to bleach bones facilitating the forensic anthropologist examination. However, prolonged exposure to chemicals often compromises DNA recovery and its amplification via PCR. Our purpose was to test the new generation multiplexes to type nuclear DNA and contemporarily to verify the possibility of mt-DNA sequencing. Our work demonstrates the ability of NGM kit (Applied Biosystems, Foster City, CA) to give a result when applied after an extraction strategy that implies large amounts of bone powder, a high affinity column separation and a "small meshed molecular size" concentration. Finally, we suggest not to use chemicals to macerate human bones for the purpose of identity or at least to save a fragment of femur from the treatment but if this is not the case, we indicate an analytical strategy that consider as the best method to obtain a nuclear-DNA profile in highly degraded DNA condition.

One hundred and fifty individuals have been sampled across Central-South Italy and genotyped for Y chromosome STRs by PowerPlex® Y system. Comparison with previous Italian databases revealed that majority of Y chromosome variation still need to be sampled. Identification of locus duplications, distribution of genetic variation and firstly identified alleles point to the necessity of more focused sampling strategies for reference databases.

Terrorist crime usually makes use of improvised explosive disposals (IEDs) which, especially in national environments, consist of postal or pipe bombs often assembled by using plastic or paper envelopes, adhesive tapes, electric components such as switches, batteries, cables, etc. To date it has been considering quite complex to recover DNA in quality and quantity proper for STRs typing aimed at the identification of the manufacturer of the IED especially due to the very serious damage degree of the evidence (exposed to high temperatures). In this work we tested saliva and touch evidences (perspiration), usually found on a postal package, deposed inside the postal bombs charged with deflagrating or detonating explosives in different quantities and we evaluated the possibility to achieve DNA profiles useable for identification purposes from such kind of post-explosion biological evidences. We demonstrated that with new generation multiplexes it is possible to gain STRs profile useful for identification purposes even from highly burned and degraded bomb debris.