Researchers from Uppsala University, Max Planck Institute for Evolutionary Anthropology, the Russian Academy of Sciences, and the University of Tuebingen have developed statistical software called postmortem degradation tools (PMDtools) that differentiates between aged DNA and contaminating sequences from more recent genetic material in samples collected from ancient bone fossils.
Contaminated samples are a problem for researchers trying to analyze ancient DNA. Research laboratories have strict protocols that they use to retrieve DNA from bone fossils and ensure the integrity of the sample. However, many of the bones used in these sorts of studies are part of decades-old museum collections or have been used in other sorts of research projects — morphology studies for instance — and, as a result, carry contaminating modern human DNA that "can impose major biases on population genetic and phylogenetic analyses," the researchers wrote in a recently published Proceedings of the National Academy of Sciences paper that describes their solution to the problem.
The team's software looks for patterns of degraded DNA caused by "miscoding lesions," the paper explains. These degradation patterns are one of several features that previous research shows is a characteristic of ancient DNA fragments. It shows up in older DNA sequences as "cytosine [to] thymine substitutions that [increase] toward the 5′ end of the sequence reads, which in most applications, results in a complementary guanine [to] adenine pattern in the 3′ end caused by enzymatic repair," the researchers wrote. It also "shows a clear tendency of increase over time in contrast to other potential diagnostic features, such as fragment length and preferential fragmentation at purines," they said.
PMDtools uses a likelihood framework "to gauge how likely a single sequence is to have originated from an ancient template molecule" based on the presence of this damage pattern whilst accounting for differences that may be due to sequence errors or biological polymorphisms, according to the PNAS paper. For each DNA fragment in the sample, "we use this framework to evaluate two competing models, of which one assumes ancient DNA degradation and the other does not, arriving at a final log-likelihood ratio of the two models [called] a PMD Score" where "a positive PMDS for a DNA fragment indicates support for the ancient DNA model relative to the alternative model," the researchers wrote.
Simply put, PMDtools aligns sequences from a given bone sample to a reference genome and looks for matches and mismatches — in terms of C to T and G to A — between the two sets of sequences, Pontus Skoglund, the study's lead author and a researcher in the evolutionary biology department at Uppsala, explained to BioInform. It then "adds all those mismatches and matches up [and] weighs them according to whether a match or mismatch is observed near the end or in the middle," where sequences that have mismatches closer to the end are more likely to come from DNA from the original bone, he said. The software generates a PMD score for each fragment. "We put a threshold on that score … [and] only keep those sequences that have a higher value than [the threshold] for further analysis," Skoglund said.
The resulting software effectively reduces contamination in ancient human DNA to "negligible levels," offering advantages over previously published methods for removing contaminating sequences including one that works by "dividing sequences based on fragment length," the researchers wrote.
Among other tests to demonstrate their method's efficacy the researchers used PMD Tools to analyze a contaminated sample of mitochondrial DNA taken from a Neanderthal bone found in the Okladnikov cave in the Siberian Altai Mountains and were able to separate the degraded DNA belonging to the Okladnikov individual from contaminants. By comparing the cleaned up sample to previously sequenced mitochondrial DNA samples collected from other fossils, the researchers showed that this particular Neanderthal was close kin to other European Neanderthals.
"There are many really interesting ancient human remains that we can rescue from severe contamination with this method," Mattias Jakobsson , a professor in Uppsala's evolutionary biology department and a co-author on the study, said in a statement. "And the method is not limited to Neanderthals — even remains of anatomically modern humans that are contaminated by modern-day humans can be rescued," he said. In one study mentioned in the paper, PMD Tools found evidence of the degradation pattern in 50- to 100-year-old DNA samples.
Furthermore, although this study focused exclusively on human data, "contamination from present day sources also affects nonhuman ancient DNA studies of pathogens, ancient microbiomes, animals, and plants" and methods like PMD Tools could become increasingly important for researchers working with these datasets, the researchers wrote