Forensic species identification: practical guide for animal and plant DNA analysis

: The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed

Lessons learned from the first European project on the integration of infectious diseases in testing services, data collection and country responses.

Despite the progress in effective treatments for HIV, viral hepatitis, tuberculosis and sexually transmitted infections (STIs), these infections remain major public health concerns across Europe. Recurring challenges of late presentation and unprioritized prevention programmes need to be effectively addressed in order to control and prevent transmission and ensure that people are diagnosed early and rapidly enter the care system. The prevalence of co-infections is high due to the social context of key populations and the shared modes of transmission, varying with local epidemiology, which underlines the need to combine efforts throughout the continuum of care

Forensic botany: species identification of botanical trace evidence using a multigene barcoding approach

Forensic botany can provide significant supporting evidence during criminal investigations. However, it is still an underutilized field of investigation with its most common application limited to identifying specific as well as suspected illegal plants. The ubiquitous presence of plant species can be useful in forensics, but the absence of an accurate identification system remains the major obstacle to the present inability to routinely and correctly identify trace botanical evidence. Many plant materials cannot be identified and differentiated to the species level by traditional morphological characteristics when botanical specimens are degraded and lack physical features. By taking advantage of a universal barcode system, DNA sequencing, and other biomolecular techniques used routinely in forensic investigations, two chloroplast DNA regions were evaluated for their use as "barcoding" markers for plant identification in the field of forensics. We therefore investigated the forensic use of two non-coding plastid regions, psbA-trnH and trnL-trnF, to create a multimarker system for species identification that could be useful throughout the plant kingdom. The sequences from 63 plants belonging to our local flora were submitted and registered on the GenBank database. Sequence comparison to set up the level of identification (species, genus, or family) through Blast algorithms allowed us to assess the suitability of this method. The results confirmed the effectiveness of our botanic universal multimarker assay in forensic investigations

Species identification through DNA "barcodes

Conventional methods for forensic species identification are mainly based on immunological procedures, which have limited applications for old and degraded specimens. The mitochondrial cytochrome b gene sequence has emerged in forensics among molecular methods. Recent investigations in the taxonomic field have suggested that a DNA-based identification system may aid the resolution of animal diversity and classification using sequence analysis and phylogenetic links. Selected gene sequences can be viewed as a genetic "barcode," which is enclosed in every cell, and barcoding is a standardized approach for characterizing species using short DNA sequences as a diagnostic biomarker for organisms. The aim of this study was to evaluate the potential of barcode mitochondrial genes, such as the cytochrome c oxidase sub 1 (COI) and the 16S rRNA gene, as a forensic tool. We developed a new approach for species testing and identification with a singleplex PCR amplification that will be useful not only in criminal casework but also in biosecurity, food authentication, investigation against poaching or illegal trade of endangered species, and wildlife enforcement. Seven fragments ranging from 157 to 541 bp (base pairs) in humans were selected from COI and 16S rRNA genes by different redesigned sets of primers suitable for forensic purposes. The specificity of each primer pair was evaluated with a single PCR reaction on different substrates, and the diversity values were calculated by statistical tests to select a set of markers that could be useful in different caseworks. A case example of forensic species identification is also presented

Slow and fast evolving markers typing in Modena males (North Italy).

One hundred thirty male individuals, strictly selected for their geographical origin and for typical regional surnames were submitted to the analysis. 17 STRs (short tandem repeats) loci and 19 SNPs binary markers (single nucleotide polymorphisms) of male-specific region of the Y chromosome (MSY) were typed to well characterize the selected population of Modena province. The availability of joint distribution of MSY haplotypes and haplogroup frequencies is becoming an important tool for both human evolutionary studies and forensic investigation, but large databases of complete Y-lineages are needed for a better understanding of the power of the combined use of Y-specific polymorphisms. A total of 129 haplotypes and 9 haplogroups were found and R1b haplogroup with a frequency of 67.7% was the most frequent, as expected because of the geographical location of the sample (Northwestern Italy). The Modena Y-lineages (STRs and SNPs independently) were also compared with published data of other neighbouring populations' samples

Two caseworks for one gene: successful species identification from compromised bone materials with the 12S rRNA

The availability of a reliable molecular assay in species recognition in forensic cases is of paramount importance when visual inspection or morphological methods are not exhaustive, especially from challenging samples. Here, two different caseworks involving bone samples founded during medico-legal outdoor investigations are presented. In order to exclude the human nature of the specimens and to determine the exact species they belong to, we proceeded with the molecular approach trying to generate sequences from the classical mtDNA markers cyt b and COI. However, they both gave critical results. For this reason, a short amplicon of ~ 150 bp of the 12S rRNA gene was used as an alternative.This short fragment was sufficient to identify the biological origin of the bone specimens with a high degree of certainty leading to the exclusion of their human nature. This work highlights the utility of the 12S rRNA and underlines the importance of deepen the choice of alternative shorter markers with respect to the classical ones, in order to achieve species identification even from challenging and degraded material in forensic criminal and wildlife caseworks

Development of six-SNPs assay for forensic analysis in European population

Y-chromosomal SNPs analysis show regional specificity useful in forensic investigation for inferring the male genetic background of individuals and population and to predict biogeographical origin of the donor of a crime scene sample. Due to its exclusively paternal inheritance, the Y-chromosome has been extensively used in evolutionary and forensic genetics to investigate the phylogeny and the history of population and their migration. A large scale parsimonious phylogenetic tree representing worldwide Y-chromosome variation has been constructed and comprises major haplogroups. The aim of this study was to set-up six multiplex assays based on SNaPshot kit to identify markers inside major clades of European population. Specifically, we design PCR and minisequencing primers targeting a total of 33 Y-mutations downstream R1*, I*, J2*, G* and E1b1b1* haplogroups. The PCR fragments were chosen to get the shortest product possible in order to improve the performance in degraded samples (amplicons principally ranging from 56 bp to 140 bp). This assay based on a 6-multiplex PCR reaction is a suitable tool for detecting the main European haplogroups in forensic casework and population study

Forensic botany II, DNA barcode for land plants: Which markers after the international agreement?

The ambitious idea of using a short piece of DNA for large-scale species identification (DNA barcoding) is already a powerful tool for scientists and the application of this standard technique seems promising in a range of fields including forensic genetics. While DNA barcoding enjoyed a remarkable success for animal identification through cytochrome c oxidase I (COI) analysis, the attempts to identify a single barcode for plants remained a vain hope for a longtime. From the beginning, the Consortium for the Barcode of Life (CBOL) showed a lack of agreement on a core plant barcode, reflecting the diversity of viewpoints. Different research groups advocated various markers with divergent set of criteria until the recent publication by the CBOL-Plant Working Group. After a four-year effort, in 2009 the International Team concluded to agree on standard markers promoting a multilocus solution (rbcL and matK), with 70-75% of discrimination to the species level. In 2009 our group firstly proposed the broad application of DNA barcoding principles as a tool for identification of trace botanical evidence through the analysis of two chloroplast loci (trnH-psbA and trnL-trnF) in plant species belonging to local flora. Difficulties and drawbacks that were encountered included a poor coverage of species in specific databases and the lack of authenticated reference sequences for the selected markers. Successful preliminary results were obtained providing an approach to progressively identify unknown plant specimens to a given taxonomic rank, usable by any non-specialist botanist or in case of a shortage of taxonomic expertise. Now we considered mandatory to update and to compare our previous findings with the new selected plastid markers (matK+rbcL), taking into account forensic requirements. Features of all the four loci (the two previously analyzed trnH-psbA+trnL-trnF and matK+rbcL) were compared singly and in multilocus solutions to assess the most suitable combination for forensic botany. Based on obtained results, we recommend the adoption of a two-locus combination with rbcL+trnH-psbA plastid markers, which currently best satisfies forensic needs for botanical species identification

vIRONy: A Tool for Analysis and Verification of ECA Rules in Intelligent Environments

Intelligent Environments (IE) are a very active area of research and a number of applications are currently being deployed in domains ranging from smart home to e-health and autonomous vehicles. In a number of cases, IE operate together with (or to support) humans, and it is therefore fundamental that IE are thoroughly verified. In this paper we present how a set of techniques and tools developed for the verification of software code can be employed in the verification of IE described by means of event-condition-action rules. In particular, we reduce the problem of verifying key properties of these rules to satisfiability and termination problems that can be addressed using state-of-the-art SMT solvers and program analysers. We introduce a tool called vIRONy that implements these techniques and we validate our approach against a number of case studies from the literature