A semi-automated protocol for NGS metabarcoding and fungal analysis in forensic.

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.

Improvement and automation of a real-time PCR assay for vaginal fluids.

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.

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.

Type 1 diabetes onset and pandemic influenza A (H1N1).

Type 1 diabetes (T1D) is a heterogeneous disorder characterized by destruction of pancreatic beta cells, culminating in loss of insulin secretion. Data from large epidemiologic studies worldwide indicate that during the last decades the incidence of T1D has increased significantly, reaching percentages of 2-5% annually. This increase suggests that there is a significant environmental contribution impacting the development of the disease, since genetic factors alone can hardly explain the rapid increase. Studies regarding T1D epidemiology in diverse populations aim to identify the disease causal factors and new targets for intervention. Viruses are one of the environmental factors implicated in the development of T1D in susceptible individuals. Recent studies suggest an association of T1D with H1N1 influenza. We would like to comment on this association and report our experience. Prospective studies are necessary to assess whether H1N1 infection is involved in T1D pathogenesis and provide directions on how to deal with viral infections in diabetes-susceptible individuals.