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.