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.

In case of severely burned bodies, victim identification by visual or fingerprints recognition is often prevented by altered body conditions. To overcome these circumstances, different techniques are available. Among these, the most reliable is molecular identification, especially in cases of detached body parts. However, DNA analysis of highly burned remains is a very challenging task. The high temperatures reached at the time of burning can lead to the complete destruction of the genetic material, making DNA typing arduous. This work presented a successful identification through molecular analysis of 11 heavily carbonized victims assigned to the Institute of Legal Medicine of Modena (Italy) between June 2022 and June 2023: a helicopter crash, a femicide and two car accidents. Post-mortem (PM) and ante-mortem (AM) data were compared, allowing victims' identification and their quick return to relatives. Complete autosomal and Y chromosome STRs profiles were obtained for all the corpses. For the helicopter crash case, the utility of the DVI module implemented in the Familias software is shown as this aid the fast association of the seven victims involved with the familiar references available for identification. The importance of the sampling strategy and the need of a systematic approach to select the most promising biological material for a more successful downstream DNA-based identification is also highlighted.

In recent years, Forensic DNA Phenotyping (FDP) has emerged as an innovative "DNA intelligence approach" aimed at predicting externally visible individual characteristics from biological traces collected at crime scenes or from biological samples of unidentified corpses or skeletal remains. This study aims to analyse the allele frequency distribution of genetic markers involved in the prediction of eye and hair colour in an Italian population sample, to enhance the existing European dataset. A large cohort of Italian individuals with at least three generations of ancestry was recruited, ensuring representation of phenotypic variability. The 24 SNP markers included in the HIrisPlex panel were analysed using SNaPshot sequencing technique, and the genotypic data were uploaded to the FDP web tool validated for forensic use by the international VISAGE Consortium's. This tool provides prediction probabilities for three eye colour categories (blue, brown, and intermediate) and four hair colour categories (black, blonde, brown, and red). The results confirmed a high predictive accuracy for the Italian population as well; however, the complex genetic structure of intermediate phenotypic traits highlights the need for novel prediction models that include genetic markers associated with complex phenotypes. The strong north-south European gradient in eye colour, along with the hypothesis that variation in eye colour is associated with skin pigmentation and UV environmental adaptation, underscores the importance for further research. These should focus on novel genetic markers to refine the European dataset, in which the Italian population may be included to enhance the accuracy of predictive models.

Recent studies focus on the different ways in which DNA can be transferred from and individual to another or to a surface. This work investigates how mixed DNA profiles can be generated on items commonly collected at the crime scene, specifically on cigarette butts. The aim is to determine how biological material exchanged during intimate contact (kissing or sharing a cigarette) can be secondarily transferred onto cigarettes, and how long non-self DNA persists and remains recoverable under different conditions and time intervals. The preliminary experimental study involved one investigative couple: each volunteer smoked a cigarette at fixed intervals after kissing (from 0 to 120 min) and both partners smoked a shared cigarette. DNA was extracted from cigarette butts immediately and after 30 days using the QIAamp DNA Investigator Kit on the QIAcube Connect. Quantification was performed with PowerQuant® System Kit and genetic profiles were obtained using PowerPlex® Fusion and Y23 Kits. Results showed non-self DNA alleles up to 2 h after kissing, even in non-immediate extractions, partial Y-STRs profiles in female butts after 1 h: individual variability and degradation made interpretations harder. Based on preliminary findings, the study will be expanded including more partner pairs, more smoking devices and longer processing timeframes. Our findings suggest the possibility of reconstructing transfer scenarios leading to mixed DNA traces on cigarette butts and of assessing a time window within which non-self DNA can be secondarily transferred to the cigarette, highlighting the impact of processing times and smoking device on DNA results.