## 4 Molecular Analyses
### DNA Analysis
#### 4.2.2 Microbiome Analyses and Applications  
Since the technological development of the next-generation sequencing (NGS) techniques, in particular metagenomics, some more comprehensive studies have been published that allow for a very complete analysis not only of the animal origin of the parchment, but also of its microbiome [2; 3; 5; 8; 20; 21]. The microbiome is the community of microorganisms (such as fungi, bacteria and archaea) and even viruses that exists in a given environment. In the specific case of parchments, it is the total community of microorganisms that has inhabited, or is inhabiting, the surface of the document. However, the question is: does a parchment have a specific microbiome? Reviewing the studies that have been carried out so far by molecular methods, we can say that the parchment possesses a core microbiome, which reveals on the one hand, the history of its manufacture and, on the other hand, the history of the use of the documents. Metagenomics using target sequencing have been applied on parchments to reveal that halophilic/halotolerant archaea and bacteria are the main players in the parchment microbiome [5; 20]. However, surprisingly, viruses have also been detected in relatively high proportions in the studies where non-targeted sequencing or the so-called \"shotgun approach\" has been used [2; 3; 8]. This last approach involves the sequencing of the total extracted DNA, without focusing on any target sequence, which allows the simultaneous identification of both the raw material (animal skin) that was used for the manufacture of the manuscript as well as the different life domains that make up the total microbiome inhabiting the object. In this context, Figure 5 shows the microbiomes that were obtained in our latest studies on the ancient Slavonic codices, which are mentioned above, by shotgun sequencing. In all of the samples that were analyzed, it was possible to determine a dominance of bacteria, an unexpected relevance of viruses and a low relative abundance of eukaryota and archaea. As Figure 5 shows, a great advantage of performing shotgun sequencing is the visualization of the microbiome members with their real proportions in the community. However, despite these advantages, to date, there have been very few studies using this approach to study the microbiomes of parchments [2; 3; 8]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Codex** & _Homo sapiens_ & _Bos taurus_ & _Ovis aries_ & _Capra hircus_ \\ \hline _Liturgiarium Sinaiticum_ S5 & 208,416 & 15,204 & **49,223** & 8803 \\ _Liturgiarium Sinaiticum_ S6 & 64,370 & **4630** & 483 & 174 \\ _Codex Assemanianus_ P & 7,852,951 & 13,544 & **55,146** & 11,576 \\ _Codex Assemanianus_ R & 143,268 & 19,958 & **24,465** & 3972 \\ _Kiev Folia_ & 5,903,087 & **356,967** & 289,404 & 347,337 \\ _Glagolita Clozianus_ & 243,594 & 180,518 & **224,188** & 59,737 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Animal species identified in investigated ancient Slavonic codices showing the total numbers of reads affiliating with each animal species. The data concerning the _Liturgiarium Sinaiticum_ and the _Codex Assemanianus_ are published in [PERSON] et al. 2020 [3], the data from the _Kiev folia_ in [PERSON] et al. [8] and the data from the _Glagolita Clozianus_ are shown here for the first time. The whole methodological and data analysis procedure that was applied to the latter codex was done in exactly the same way as it was for the Kiev Folia [8] (for more details see supplementary Material File S1 and Figure S1). Bold marked: indicate the highest number of reads related to a specific animal besides humans in each sample. The prokaryotic community that has been identified so far by molecular approaches in the parchment microbiome includes members that can be grouped into two main groups: those that are typical of a saline environment and those that are typical of the human microbiome. The first group comes into contact with the parchment at a very early stage, i.e., during the manufacturing process [5; 20; 21; 63].