## 4 Molecular Analyses
### DNA Analysis
#### 4.2.1 Identification of Animal Skins via DNA Analyses  
One of the main applications of the genetic analysis of parchment is, and has been in the past, to discover the origin of the skins that were used for its manufacture. This involves the isolation of the ancient DNA (aDNA) that may still be preserved in the parchment. However, the alteration of the material throughout its history, including during its manufacture, preservation and analysis, affects the endogenous DNA of the parchment from the time when the skin was removed from the donor animal until it is extracted in the laboratory for the DNA analysis several hundred years later. When an organism dies, its DNA is generally degraded by endogenous nucleases, but chemical damage, such as oxidation or hydrolysis, also occurs. These processes lead to breaks in the DNA strands, the loss or alteration of the bases in the nucleotides, and the cross-linking of the strands [44]. Furthermore, microbial attack also contributes to the breakdown of the DNA molecules. However, it may be the case that under certain favorable circumstances, such as a low temperature, rapid desiccation, or high salt concentrations, the nucleases themselves may be destroyed or inactivated before all the nucleic acids are degraded [57; 58; 59]. The processes that were used in the manufacture of the parchment include some treatment conditions that may have allowed some of the DNA in the skin to remain intact [44], as previous research has suggested that both the mitochondrial and autosomal DNA may be well preserved in this material [1; 45]. In fact, there is a compilation of research showing that ancient DNA can be detected in a variety of museum artefacts as well as biological and archival materials, including parchment [60]. As mentioned above, some of these studies were conducted before the NGS platforms were available and they used PCR-based assays using mitochondrial and/or nuclear markers for the identification of the animal skins that were used in the production of the parchment [46; 48; 50; 61]. However, several shortcomings that are related to PCR-based assays have been documented, such as the control of the contamination risk, the high proportion of PCR chimeras and the questionable results that are obtained with the cross-contamination between individuals and species [48; 61]. In addition, PCR facilitates the amplification of longer and less-damaged DNA fragments, thus it favours the modern-contaminating DNA over endogenous aDNA [1]. Next-generation sequencing (NGS) approaches allow a very thorough analysis of the ancient parchments to infer the proportion of the endogenous DNA (species of origin) that is retained in the different samples as well as its possible contamination with other animal species. [PERSON] et al. [1] performed a first shotgun NGS study on parchment, and they showed that this material is a very usable substrate for aDNA recovery and analysis. They were able to retrieve 7-9% of the endogenous ovine genome from two of the investigated parchment documents dating from the 17 th and 19 th centuries at a high-quality and estimated-to-be-comparable or an even lower external contamination rate than that which was obtained from aDNA that was extracted from other materials. They assumed that the alignment rates of the non-source species were probably due to the homology between the genomes of the ruminants, as well as the damage that occurred to the aDNA molecules, resulting in a higher alignment with the non-source species. In a further study, [PERSON] et al. [2] investigated samples that were derived from the 1000-year-old bifolia of the York Gospels, and they estimated that there was an average percentage of endogenous DNA of 19.3% (range 0.7-51.4%) in all of the samples. However, these calculations fell in the range of 0.2-5.7% when filtering for the quality of the mapping reads was applied. Later, [PERSON] et al.