Innovative approach to study the Biocodicology of parchment manuscript

The study of the ancient manuscript, and in particular the investigation of the biological information stored in it, takes the name of “Biocodicology” and it allows to study ancient manuscript in different ways [1] such as microscopic and visual analysis, or high-throughput molecular techniques like proteomics [2], genomics and metagenomics analysis [1], [3], [4]. In particular, the use of biomolecular techniques in recent years, allowed us to do analyses that are unthinkable a few years ago [5] the study and manipulation of DNA allow us to get two types of information: diagnostic of biodeterioration through the identification of fungi and bacteria species that degrades the artifact [6]–[8], to intervene with targeted restoration and conservation interventions; obtain information about the composition of the material, such as the skin animal species with which the parchment was created, both to recreate the skin for possible restorations and to reconstruct, through a phylogenetic study, the place and period in which the parchment was created [9]. The major challenges in this field are the realization of non-invasive sampling [10] and the manipulation of small and fragmented ancient DNA (aDNA). In this study we develop a workflow that provides: 1) innovative, non-invasive sampling methods (swab and PVC eraser) already used in the field of restoration and in recent works [2], [4], [11] that is a waste product from the cleaning process; 2) DNA extraction with the CTAB buffer; 3) DNA amplification with rDNA kingdom- specific primers for fungi (ITS), and bacteria (16S) to analyse the biodeterioration, and with primer based on the conserved region of cytochrome b (mtDNA) of Capra hirus, Ovis aries and Bos taurus to discover the animal species of parchment, 5) sequencing with Nanopore technologies and 5) Data analysis through a customized bioinformatic pipeline, with Fungi and Bacteria database for the Biodeterioration and Eukaryota database for the animal species. The workflow was tested with good result: -on ancient parchment, for the identification of biodeterioration, allowing us to identify different fungi species like Cladosporium, Alternaria alternata and others usually find it in parchment material, and bacterial species like Staphylococcus epidermidis, and Cutibacterium acnes linked to the human presence; and -on a modern parchment for the identification of animal species, which turned out to be made of Bos taurus skin. The next step is to test the complete workflow in a real case study, i.e., on a PVC eraser sample taken from ancient parchment, for the identification of fungi, bacteria, and animal species within a single analysis to speed up the workflow. Non-invasive sampling, the use of PCR for the amplification of DNA, and the Sanger and Nanopore sequencing methods, resolve in part the problem and allow to use of this analysis routinely in subsequent works.