## 4 Molecular Analyses
### DNA Analysis
#### 4.2.2 Microbiome Analyses and Applications
Within the phylum _Frimicutes_, mainly the species of the genera _Staphylococcus_ and _Streptococcus_, but also _Leuconostoc_, _Lactobacillus_ and _Gemella_ as well as the members of the family _Carnobacteriaceae_ have been identified [2; 3; 8] (and this study, see Figure 7). It is important to highlight that most of the human microbial markers that have been found on parchments are characteristic of the microbiome of the skin and the oral [8] and nasal cavity [2], which points to the history of use of the parchments that have been investigated. The bacteria that were detected not only indicate the intensive handling of the parchments over the centuries, but also to the usage and devotional customs and habits of the past, such as the custom of kissing prayer books during religious ceremonies, which would explain the large number of bacteria that are typical of the oral cavity being present on the surface of religious books [8]. Nevertheless, these data should be taken with caution, as many of these bacteria, on the one hand, are potential human pathogens and, on the other hand, they may also be involved in the deterioration of the parchment. To name a few examples, it has been suggested that some _Staphylococcus_ and _Streptococcus_ species may be involved in the formation of calcium oxalates on parchment [8]. Additionally, the members of the genus _Brevbacterium_ have been repeatedly detected in the parchment microbiome [2; 3; 69] (and this study, see Figure 7). Although the identification of them at species level was not always possible, in some cases _B. linens_ was detected [3]. This species is abundant on human skin, but it is also well known for its ability to tolerate high salt concentrations. In addition, it produces carotenoid pigments and proteases, which are capable of damaging parchment [79], all together, posing a severe risk to the integrity of the parchment documentary material. Eukaryotic Community in the Microbiome  
The most recent studies using shotgun metagenomics have shown that eukaryotic microorganisms represent only a small proportion of the microbiome (typically below 2%) of parchment [2; 3; 8] (and this study, see Figure 5). This observation contrasts with those of the previous studies in which only some target regions of DNA were amplified and sequenced (185 rRNA and/or ITS sequences for the target amplification of fungi) to investigate the specific groups of microorganisms that are on the parchment [17; 51], highlighting the greater accuracy of the shotgun approach to study the actual proportions of the different domains of life in the microbiomes. As an example of the application of metagenomics with a shotgun approach, Figure 8 shows our previous and current studies on the ancient Slavonic manuscripts. The figure reveals that the small proportion of eukaryotic microorganisms in the microbiome of the investigated parchments are related, in most of the case studies, to the phylum _Ascomyca_, mainly to the families _Aspergillusaceae_, _Debaryomicetaceae_ and _Saccharomycesectaceae_[2; 3] (and in this study, see Figure 8) and they are specifically affiliated to sequences of the genera _Penicillium_, _Debaryomyces_ and _Saccharomycesces_, respectively (data not shown). In addition, the members of the phylum _Basidiumyocta_ have also been identified in three of our case studies [3; 8], (and in _Glagolita Clozianus_ in this study), with all of them being related to the _Malassezia_ species. Finally, in two of the manuscripts that we have investigated, the _Eimeria_ species belonging to the family _Eimeridae_ (phylum _Apicomplexa_) were identified instead [3]. Despite the low overall proportion of the eukaryotic microorganisms that were found in these ancient Slavonic manuscripts, the data that were recovered may also provide some interesting insights into the raw material that was used for the manufacture of some of the parchments which were investigated. For example, some of the sequences that were identified in two of the Slavonic manuscripts (_L. Sinaiticum_ 5 and _C.