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Paleogenetic extraction and analysis from challenging environments

Periodic Reporting for period 1 - PACE (Paleogenetic extraction and analysis from challenging environments)

Reporting period: 2016-06-01 to 2018-05-31

This project’s overarching aim is to study ancient DNA preservation in challenging environments, particularly from archaeological collections from warm and humid or warm and arid environmental contexts. In this way, we are looking to increase DNA yield from samples that normally do not contain a large amount of endogenous DNA (DNA specific to the individual sample). We chose to focus on a wide temporal span, from as early as the Neolithic (~5000 years before the present, yBP) to 800 yBP. Our research focuses on the human petrous bone as this has consistently shown to have a higher amount of endogenous DNA compared to other bones in the human skeleton. Our sampling approach consists of targeting the inner osseous canal, consisting of the cochlea and semi-circle canals. We use advanced DNA extraction and next-generation library sequencing methods to obtain our stated goals. This research is important for the ancient DNA community as it attempts to expand our understanding of how humans interacted and evolved in different regions of the world. Our overall objectives are to create more efficient sampling protocols to achieve our stated goals.
The Fellow sampled from an existing collection of archaeological samples at the University College Dublin, Ireland and sampled on site in western Ukraine. Samples, human petrous bone, were used to infer differences in endogenous DNA yield from warm and humid or warm and arid environments, which are particularly challenging for researchers in the field of human ancient DNA. The reasons for the lack of quality DNA from these environments are varied, ranging from soil pH to burial context. We employed a sub-sampling approach to the inner osseous canal of the petrous bone, as this has consistently shown to yield higher amounts of human DNA compared to other skeletal elements. However, researchers have only been able to obtain small (less than 1%) of endogenous DNA from these samples; or use expensive technologies such as in-solution or array-based target capture to obtain higher yields from these difficult samples. We developed a protocol that targets several regions within the inner osseous canal that we hope will benefit future researchers who work with such samples. Preliminary results show that the most successful region for obtaining quality DNA is the cochlea; however, intra-sample and intra-site variability make observing consistent patterns difficult. Therefore, we suggest a case-by-case basis for archaeological samples deriving from these environments. Our sampling approach has yielded high endogenous DNA that will bring down the costs for future researchers. The Fellow undertook several experiments in parallel with his sub-sampling strategy that implemented how NGS library techniques might influence sequencing complexity and endogenous DNA yield. To this end, he constructed over 500 NGS libraries, of which over 250 were sequenced on Illumina platforms. Specifically, he prepared libraries according to a traditional approach, a modified traditional approach, and a technique called the single-stranded method that has successfully been used to sequence Neanderthal and Denisovan samples. The single-stranded method has also been shown to work well for highly degraded samples of which the Fellow was working with. To this end, he built several libraries according to these different methods from the same extract and found that only the most degraded samples benefit from the single-stranded approach. This is important, as the single-stranded approach is costlier than the more traditional double-stranded methods and more time consuming. Therefore, for samples that are relatively of good quality, the less expensive and higher throughput double-stranded approach is best.
Only recently have researchers been successful in obtaining genome-wide data for archaeological samples that derive from challenging environmental contexts. This project went beyond the traditional sampling approach and attempted to use state-of-the-art next-generation sequencing technologies to achieve it’s goal of being able to develop protocols for other researchers to benefit from. The world of human ancient DNA is costly. Although sequencing costs have come down in recent years, the cost of sequencing a sample that yield little or no DNA are not economically feasible. Therefore, our project aims to bring down the cost of sequencing by initially sampling in the correct location in order to optimise DNA yield that will make difficult archaeological samples viable for population-level analyses.