Improved detection of methylation in ancient DNA

Abstract

Reconstructing premortem DNA methylation levels in ancient DNA (aDNA) has led to breakthrough studies such as the prediction of anatomical features of the Denisovan, as well as the castration status of ancient horses. These studies relied on computationally inferring methylation levels from damage signals in naturally deaminated cytosines. Because of statistical constraints, this inference requires high-coverage sequencing, and is thus not only expensive but also restricted to samples with exceptional DNA preservation. Instead, a method to directly measure methylation levels in aDNA, as exists in modern DNA samples, would open the door to a more thorough and cost effective ability to study ancient DNA methylation. We have tested two methods for direct methylation measurement developed for modern DNA based on either bisulfite or enzymatic methylation treatments. We find that both methods preserve sufficient DNA yields to allow for methylation measurement. Bisulfite treatment, combined with a single stranded library preparation, shows the least reduction in DNA yields compared to no methylation treatment, as well as the least biases during methylation conversion. In addition, we show that applying bisulfite treatment to ∼0.4-fold coverage sample provides a methylation signal that is comparable to, or even better, than the computationally inferred one. We thus present a method to directly measure methylation in ancient DNA that is cost effective and can be used on a wide variety of ancient samples.

Investigating food production‐associated DNA methylation changes in paleogenomes: Lack of consistent signals beyond technical noise

“…Meanwhile, using alternative experimental strategies could also be an option for paleomethylomics. For instance, Sawyer et al (2023) recently reported that bisulfite treatment of aDNA coupled with a single-stranded library preparation protocol could produce similar or even more accurate CpG methylation estimates as that inferred indirectly from postmortem damage, using much lower amounts of sequencing data.…”

“…However, it has been shown that cytosine methylation sites can survive in ancient DNA. Several studies have used standard protocols for methylation profiling, such as bisulfite sequencing and immunoprecipitation, on ancient DNA (Llamas et al., 2012 ; Sawyer et al., 2023 ; Seguin‐Orlando et al., 2015 ; Smith et al., 2015 ). Meanwhile, methylation information can be indirectly inferred from sequencing data from ancient DNA molecules treated with the UDG (uracil‐DNA glycosylase) enzyme.…”