Bisulfite Sequencing of Ancient DNA May Lead to Clues about Old Populations

Bisulfite Sequencing of Ancient DNA May Lead to Clues about Old Populations

For the first time, anthropologists from The University of Texas at Austin have shown that epigenetic tags on DNA, such as DNA methylation, can be successfully detected in the remains of ancient human DNA using bisulfite sequencing. These results can help progress future studies to enhance our understanding of disease and famine experienced by those from ancient times.

Epigenetic research assesses various chemical marks made to DNA which do not alter the underlying genetic code, but impact how certain genes may be expressed. DNA methylation, for instance, can typically suppress gene expression by the addition of a methyl group (CH3) on the 5’ position of cytosine. These marks occur naturally throughout one’s life and are necessary for normal development but the epigenetic modifications may also occur as a result of environmental factors such as diet, disease, and climate. Additionally, if the epigenetic modification occurs to one’s egg or sperm DNA, these epigenetic tags could be transmitted across generations.

According to lead author and UT Austin researcher in anthropology, Rick Smith, investigating epigenetic marks allows us to “better understand what genes are expressed during a person’s life and how different environmental stresses shaped physical traits and health across generations.”

Epigenetic changes have been previously uncovered by researchers looking at the DNA of modern day humans who endured famine in utero at the time of World War II. These tags were associated with changes to diet, metabolism, and growth. Other recent epigenetic studies indicate that certain epigenetic mechanisms may be tied to the development of cancer and the progression of various diseases. By more closely investigating these epigenetic tags found on ancient DNA (aDNA), specifically human aDNA, we may be able to gain a more comprehensive view of the health and lifestyle of humans from the past.

Along with UT Austin Associate Professor of Anthropology Deborah Bolnick and Washington State University anthropologist Cara Monroe, Smith was looking for DNA methylation marks in 30 ancient human bone remains from archaeological sites located in five areas in North America. These remains ranged from 230 to over 4,500 years old. Because it has previously proved extremely difficult to recover DNA methylation or epigenetic marks in ancient DNA samples, it was a surprising find that this group was able to identify methylation in 29 of the samples using bisulfite methodologies.

Before this research, cytosine methylation was detected in DNA from one Neanderthal and one Denisovan from Serbia ranging from 50,000 and 130,000 years old, a Paleo-Eskimo from Greenland that was 4,000 years old, and a bison from Canada that was 26,000 years old. Scientists have also detected methylation in 200 and 2,800-year-old barley DNA from Egypt.

Smith and his colleagues set out to use a technique known as direct bisulfite sequencing in order to detect cytosine methylation from ancient human bone remains. According to the researchers, earlier studies have often identified epigenetic marks in ancient, degraded DNA by inferring methylation patterns “using post-mortem damage to cytosines as a proxy for cytosine methylation levels.” But this method is clearly limited in its ability to precisely assess DNA methylation, although it may be preferred when trying to avoid further degradation of especially rare aDNA samples.

Direct bisulfite sequencing is a “gold standard” method that countless epigenetic researchers utilize to investigate methylation of DNA at single nucleotide resolution and it offers a much more precise measurement. Many researchers believe bisulfite sequencing is unable to yield successful results when applied to older samples because the bisulfite conversion process can further degrade the delicate DNA due to the harsh chemical reaction involving sodium bisulfite, which converts unmethylated cytosines to uracils.

Typical degradation of old samples due to the amount of time since death and depositional conditions cause DNA damage which complicates and often misrepresents the true methylation status of aDNA. However, according to the research team, they have demonstrated the success of bisulfite sequencing applied to ancient DNA samples if these samples are more recent or better-preserved, specifically if there is “adequate preservation of endogenous nuclear DNA.”

These results prepare researchers for conducting future in depth, targeted analyses of ancient DNA using bisulfite sequencing which would help us gain additional insight into past lifestyles, disease, and diets of ancient humans. In light of this study’s successful bisulfite sequencing of preserved nuclear DNA samples more than 4,500-years-old, these results offer the first evidence that cytosine methylation can be recovered in aDNA samples. These promising findings using bisulfite techniques open new doors for a closer look into ancient methylomes at single nucleotide resolution.

In addition, the researchers reported that “precision of cytosine methylation estimates is inversely correlated with aDNA preservation” and samples with high DNA concentration displayed lower levels of variability in methylation determination. In this instance, the researchers noted a DNA concentration above 0.015 ng/μL was most desirable and produced the most accurate and consistent reading of DNA methylation status.

“By studying methylation in ancient DNA from archaeological populations, not just isolated samples, we may gain insights into how past environments affected ancient societies,” Bolnick explained. “Future research in ancient epigenetics should open a new window into the lives and experiences of people who lived long ago.”


Source: Rick W. A. Smith, Cara Monroe, Deborah A. Bolnick (2015). Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing. PLOS ONE, 10(5). DOI: 10.1371/journal.pone.0125344

Reference: University of Texas at Austin. Ancient DNA May Provide Clues into How Past Environments Affected Ancient Populations. UT News. 4 June 2015.

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About Bailey Kirkpatrick 164 Articles
Bailey Kirkpatrick is a science writer with a background in epigenetics and psychology with a passion for conveying scientific concepts to the wider community. She enjoys speculating about the implications of epigenetics and how it might impact our perception of wellbeing and the development of novel preventative strategies. When she’s not combing through research articles, she also enjoys discovering new foods, taking nighttime strolls, and discussing current events over a barrel-aged sour beer or cold-brewed coffee.


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