For PALEOS, we focused on mitochondrial DNA sequences of different domesticated animals and Y chromosomal sequences of horses. Although, these sequences are identical in individuals of the same species, there are a few differences distributed across the sequence. These differences, known as single nucleotide polymorphisms (SNPs) work as informative indicators of the specific groups called haplogroups. These haplogroups show temporal phylogeographic distribution of maternal and paternal lineage of any species. This makes it important to conclusively know to which haplogroup an individual from whom the ancient DNA is obtained, belongs. Here, we successfully designed RNA-probes for the most economically relevant domesticated animals, namely: horses, donkeys, pigs, cattle, goats, sheep, dogs and chicken. These probes target SNPs of different haplogroups of these animals. We tested the efficiency of these probes on different samples which included ancient DNA extracts obtained from diverse horse fossils. Such positive controls established the sensitivity and specificity of our in-solution DNA capture system to increase coverage by magnitudes beyond 1,000x, relative to shotgun sequencing. We further demonstrated that these probes provide sufficient power to detect the presence of different domesticated animals when applied on the DNA extracted from soil belonging to the local farm harboring different domestic animals. Our system can, thus, be used in routine for a genetic monitoring of domesticated animals present in any given environment, in the absence of invasive animal sampling; it is, thus, respectful of the animal welfare. After successful testing of probes for accuracy and efficiency, we applied this tool on ancient sediments and coprolites (~40,000 years old). Sediments were obtained from the paleolithic rock shelter Abrigo de la Malia located in the Iberian Peninsula (the region of interest), which showed early human occupation and the presence of several other mammals as well as birds. Our tool detected the presence of DNA originating from sheep in sediments deposited during past few decades when cave was being used by the farmers as a storage facility for their equipment and livestock. The mitochondrial sequences grouped with haplogroup B which is the haplogroup for domesticated sheep with ubiquitous distribution in the Mediterranean and middle Europe. This demonstrated the capacity of our methodology to detect the presence of domestic animals in the region. Applying this methodology to coprolites obtained from the cave Cassenade, France, we detected the presence of one horse maternal lineage in one coprolite, alongside those of local cave hyenas showing that the cave hyena fed on a horse before defecating in the cave. Overall, our experiments successfully demonstrate that the tool developed by PALEOS can be applied on wide variety of environmental samples. These results were presented in two conferences: The Animal Farm Meeting, Toulouse 11-13th June, 2022 and 10th International Council of Archaeozoology- Archaeozoology, Genetics, Proteomics & Morphometrics (ICAZ-AGPM) meeting, Munich. Furthermore, two manuscripts are exclusively dedicated to describe the design and the results of these probes.
