## 4 Molecular Analyses
### DNA Analysis  
DNA analyses have been applied for several years to recover the biological information that is contained in the ancient manuscripts on the parchment [44]. These analyses have been gradually developed to minimize the biases that are associated with destructive sampling in order to recover the genetic material that is contained in the parchment. Nevertheless, the amount of DNA that can be extracted from the parchment is generally low, and it may be degraded for a variety of reasons, such as the chemical processes that occur during its manufacture, the age of the parchment and the various alterations it may have undergone over the centuries due to the human manipulation or poor preservation of it. Several scientific publications have addressed this issue and all of them point to the need to develop accurate non-destructive and non-invasive sampling methodologies and precise DNA extraction protocols, as well as a careful DNA manipulation to avoid the occurrence of external contamination [1; 2; 45; 46; 47; 48; 49]. It is important to highlight that the field of biocodicology has experienced a unthinkable advance thanks to the development of next-generation sequencing (NGS) approaches, thus eliminating the intrinsic limitations of the methodologies that were used before NGS platforms became available. However, even before NGS analysis was widely used, numerous studies already highlighted the possibilities that are offered by genetic analysis of parchment [44]. Some of these studies focused only on ancient DNA (aDNA) for the sole purpose of identifying the animal skins that were used in the parchment production [46; 48; 50]. In contrast, other studies have focused on the microbiological research of manuscripts to identify the risk of potential damage that is exerted by microorganisms [17; 18; 51; 52] as well as to establish restoration/conservation methods [53]. Nowadays, advances in non-invasive sampling techniques, together with the development of NGS approaches, are making it possible to investigate the genetic material of the parchment on a larger scale. These analyses are still far from being routinely applied on ancient parchment due to the laborious work that is required for sequencing techniques, but also due to the high price of such analyses, which can only be afforded within special research projects. Nevertheless, there are already some studies in which high-throughput NGS analyses coupled with bioinformatics have been applied to parchment manuscripts, thus allowing the researchers to identify the aDNA [2]. The compilation of these data has resulted in a specific microbiome for each investigated parchment or \"bio-archive\" [3; 8; 54], which can be considered to be a historical added value. The recovered biological information offers new possibilities to answer two main questions: (a) the type of skin/animal that was used to produce the parchments, and (b) the composition of the microbiome that is colonizing their surface, but also other relevant questions that are related to the manufacturing process of the book, its state of preservation and its historical use as well as the animal breeding history [1; 2; 3; 5; 6; 8; 20]. The workflow to answer these questions includes several steps that can be summarized as the use of non-invasive or minimally invasive sampling techniques which are followed by the DNA's extraction, obtained directly from the sample material. This is a very delicate and difficult task that requires experience and care to be taken. There are strict criteria to ensure the reliability of the aDNA results. There are critical conditions for which the DNA extraction procedures can occur, such as it being performed in dedicated laboratories using special devices and equipment, and there are downstream steps that will depend on the selected sequencing platform.