Ancient DNA (aDNA) extracts are often characterized by low amount of focal (endogenous) DNA, caused by degradation and contamination by DNA of exogenous origins. We thus performed extensive experiments to adapt and optimize protocols for enriching the DNA libraries for fragments of interest and, therefore, focus sequencing efforts and resources on relevant DNA targets. The method, called hyRAD (hybridization RAD), previously used mostly for museum samples of less than 100 years old, was originally by Tomasz Suchan when he was a PhD student. It allows inexpensive production of molecular probes from fresh DNA, that allow selective enrichment of the aDNA of interest. The main modification introduced consisted of the synthesis of RNA probes, as opposed to the DNA probes produced by the original protocol and was proved to decrease contamination risk, enable producing the probes at larger scales, and increase capture efficacy. The protocol has been tested with samples of varying endogenous DNA content, preservation contexts, and age, in order to establish the method suitability to the broader range of samples, and obtained excellent enrichment rates in samples with low endogenous DNA content.
As the host lab was the first to report that aDNA methylation patterns can detected in aDNA, we have also tested the potential of using methylation sensitive and non-sensitive restriction enzymes for selective targeting unmethylated parts of the genome. The probes obtained with methylation-sensitive enzyme were shown to capture more fragments that mapped uniquely on the genome and showing lower post-mortem damage rates. This result marked an important step forward for the selective characterization of genomic regions with different methylation patterns and reducing the sequencing error rates in aDNA studies.
Another important breakthrough was to characterize how the hyRAD could be applied to more distant species. We prepared molecular probes from horse and donkey (that diverged around 4-4.5 mya), used to capture aDNA libraries of horses, donkeys, and mules (which are the offspring of a horse mare and a donkey jack). We then carefully analyzed if capturing the libraries of one species against the probes produced with another one introduces any sort of allelic bias, such as for example increasing or decreasing the chances of detecting target alleles. Our preliminary analyses show, that the hyRAD does not introduce significant bias for the captures performed on the probes prepared from relatively close relatives, which gauges for the method’s future popularity and utility in deeper phylogenetic scales. Additional analyses are being finalized and the submission of the article is planned for mid-May 2021.
We have also bioinformatically screened previously obtained ancient horse genomes for the presence of pathogens, despite considerable sequencing efforts (over 10 billion read pairs), we could not identify the presence of Yersinia pestis in the data. We thus used target-enrichment procedures to test for the presence of the pathogen in horse samples. Unfortunately, none of the horse tested supported the presence of the pathogen. However, the absence of a proof cannot be confused as the proof of the absence, as the lack of Y. pestis DNA in our data could, also indicate limited detectability in our assays. Thus the negative results obtained could not be used conclusively at that stage.
One of the aims of the project was to identify the center of origin of the domestic horses. We have obtained and sequenced crucial samples, that filled the important spatial (Central-Eastern Europe) and temporal (Eneolithic/Bronze Age) gaps in the host lab dataset of ancient horse genome sequences. The results of the abovementioned analyses are included in an article covering the origin of horse, authored by more than 100 specialists in many fields. The article solves the question of the temporal and geographic origins of horse domestication, and has strong consequences for our understanding of human mobility during the third millennium Before Common Era.
Overall, the work will be published in three scientific articles presenting: 1) the first application presenting the optimizations of the hyRAD protocol for aDNA from osseous remains several thousand years old; 2) second methodological paper assessing the potential biases of the method; 3) identifying the domestication centre of the horse, underlying the spread of the domestic horse in the third millennium BCE. Additionally, the effects of the project will disseminated during at the 9th International Symposium for Biomolecular Archaeology in June 2021, the most important conference in the field. For communicating the results to a general audience, the outreach activities took the form of lectures and lab visits organized, including within Fête de la Science events in 2019 and 2020.
