Density separation of petrous bone powders for optimized ancient DNA yields

Density separation is a process routinely used to segregate minerals, organic matter, and even microplastics, from soils and sediments. Here we apply density separation to archaeological bone powders before DNA extraction to increase endogenous DNA recovery relative to a standard control extraction of the same powders. Using nontoxic heavy liquid solutions, we separated powders from the petrous bones of 10 individuals of similar archaeological preservation into eight density intervals (2.15 to 2.45 g/cm(3), in 0.05 increments). We found that the 2.30 to 2.35 g/cm(3) and 2.35 to 2.40 g/cm(3) intervals yielded up to 5.28-fold more endogenous unique DNA than the corresponding standard extraction (and up to 8.53-fold before duplicate read removal), while maintaining signals of ancient DNA authenticity and not reducing library complexity. Although small 0.05 g/cm(3) intervals may maximally optimize yields, a single separation to remove materials with a density above 2.40 g/cm(3) yielded up to 2.57-fold more endogenous DNA on average, which enables the simultaneous separation of samples that vary in preservation or in the type of material analyzed. While requiring no new ancient DNA laboratory equipment and fewer than 30 min of extra laboratory work, the implementation of density separation before DNA extraction can substantially boost endogenous DNA yields without decreasing library complexity. Although subsequent studies are required, we present theoretical and practical foundations that may prove useful when applied to other ancient DNA substrates such as teeth, other bones, and sediments.

Automated summary

Density separation is used to extract DNA from archaeological bone powders, yielding significantly higher amounts of endogenous DNA compared to standard extraction methods. By using non-toxic heavy liquid solutions, the bone powders were separated into different density intervals. The intervals of 2.30 to 2.35 g/cm(3) and 2.35 to 2.40 g/cm(3) provided the highest yield of endogenous DNA without reducing library complexity. This method can be implemented without additional equipment and extra laboratory time, making it a valuable technique in ancient DNA research.