Oxford Nanopore Technologies for Biocodicology: new strategy to study endogenous and exogenous DNA from parchment

Biocodicology, a term coined by Sarah Fiddyment in 2019[1], is an emerging field which studies the biological information stored in codex (parchment manuscripts): the microbiome, fungi and/or bacteria species that colonise the surface of the artefacts, and the animal species used for the manufacturing of the parchment. The correct identification of these two types of information allows us to intervene with a correct restoration/consolidation methodology, such as microclimate control (ad hoc for the fungi/bacteria species identified) or can give us information about the material, place and time when the parchment was created adding important details on an unknown artefact [3]. The analyses usually used in this field are visual analysis, protein analysis, or DNA analysis with Illumina Technology, which allows the sequencing of long and short fragmented DNA from parchment. Objectives This work aims to find an alternative strategy to obtain all the biological information from parchment manuscripts (endogenous and exogenous DNA) through a methodology that combines non-target Whole Genome Amplification (WGA) using Phi29 polymerase and subsequent DNA sequencing using the MinION device from Oxford Nanopore Technologies. To test the feasibility of this approach, parchments from different periods were analysed. Methodology and Results Several parchments, from the present day to the 15th century, some of them from the Graphic Collection of the Academy of Fine Arts, Vienna, were analysed. Small pieces of the parchments (in Figure) were subjected to DNA extraction followed by Whole Genome Amplification (WGA) and sequencing with the device MinION (MK1C) from Oxford Nanopore Technologies using the new Flow Cells (R.10) and the new Short Fragment Mode (SFM). The bioinformatic results show that the parchment microbiomes are composed of typical human skin flora, with bacteria belonging to species of the genera Acinetobacter, Moraxella and Staphylococcus as well as fungi (i.e. genus Malassezia), but also to halotolerant species, such as S. equorum. More importantly results enable the identification of the animal species used for the parchment in any analysed case. Conclusion This innovative molecular study shows for the first time the potential of whole genome amplification (WGA) and MinION (ONT) in the field of biocodicology, for the identification of the microbiome (exogenous DNA) and the animal species (endogenous DNA) from parchment material, indicating the feasibility of this technology not just for fungi and bacteria, but also for short fragmented and degraded endogenous DNA in parchment.