A new way to decipher the past with DNA from parchment pages

At NC State University, an English professor is searching for clues from the past. He’s looked back as far as the eighth century, knowing that lessons learned from medieval texts could benefit modern civilization for many years to come.

Unlike the typical humanities researcher, however, Tim Stinson explores beyond merely what’s written on centuries-old parchment documents. He can also learn from the DNA that’s present both within and on the materials themselves. Stinson has been leading interdisciplinary research projects for the better part of the last decade, working hand in hand with scientists to analyze any biological material that’s still salvageable after all those years.

Now, thanks to the help of three colleagues in NC State’s College of Veterinary Medicine (CVM), Stinson has a new way to collect DNA from pages of parchment—that’s faster and easier on both the DNA collector and the documents themselves.

“We’re bringing together very different disciplines—medieval and manuscript studies from humanities and an array of techniques from the sciences—to see what information we can glean genetically to contextualize the written data,” says Stinson, an associate professor of English and University Faculty Scholar in the College of Humanities and Social Sciences.

Researchers have known for a while that our ancestors’ writing surfaces might hold more information than merely what’s inscribed on them. But collecting DNA from materials degraded by harsh manufacturing processes, Mother Nature and Father Time—not to mention handled repeatedly—is no small feat.

Above all, the problem is these artifacts are precious—and, understandably, the organizations responsible for their preservation have strict rules about how to handle them, to ensure you leave no trace. Collecting DNA from these documents without anyone being able to tell you touched it, even under a microscope, poses “a very tricky forensics problem,” Stinson says.

Until recently, an eraser was the only way to collect cellular material without causing damage. By gently rubbing a PVC-based eraser on an artifact, the eraser crumbs pick up cellular material through static electricity. You can then collect DNA from that cellular material. But as you might imagine, the eraser-crumb collection method takes a lot of time—and physical effort.

“It took me two full days of work to test one book thoroughly,” Stinson says. “And it’s tiring. I basically got tennis elbow—my arm was really sore afterward.”

So Stinson came to his CVM colleagues with a novel idea for a noninvasive collection technique.

Stinson worked with CVM faculty members Matthew Breen, Ben Callahan and Kelly Meiklejohn to validate a brand-new way to carefully collect cellular material containing DNA from documents that have been preserved for hundreds of years.

“Knowing this technique could work from our pilot testing, we wanted to test it on a broad range of samples in the Duke University’s David M. Rubenstein Rare Book & Manuscript Library,” says Meiklejohn, an associate professor in CVM’s Department of Population Health and Pathobiology. “In collaboration with Duke, we collected 300 or so samples from a range of documents.”

The research team proved cytology brushes—specifically, those used to test for cervical cancer—could collect cellular material containing DNA from documents dating back to 700 C.E. without causing any damage. Stinson says they showed their brushing technique, coupled with new sequencing methods, can capture whole mitochondrial genomes, which in turn, are used to determine source species. They published their findings in the journal PLOS One last March.

Not only is collecting material with cytology brushes far faster and easier than collecting eraser crumbs, but it also greatly reduces the risk of cross-contamination. If you’re testing an entire book with an eraser, for instance, you must ensure to get up all the crumbs from one page before starting another.

“Otherwise, you risk mixing them together,” Stinson says.

Plus, from a library’s perspective, using a brush can in some cases be even less invasive than using an eraser. Many manuscripts that have survived hundreds of years aren’t exactly clean. Erasers remove dirt or stains—and leave unsightly white spots. Stinson and his team’s collection technique does not.

What we can learn

Long before humans discovered DNA, we learned how to breed domesticated animals selectively for desired genetic traits—better wool, for example.

“But we didn’t really have any way of looking back in time to see where these breeds of domesticated animals came from,” Stinson says. “Well, now we’re hoping that we do.”

That’s just one example of how collecting DNA from historical documents can complement the information written on them to paint a more complete picture of the past.

“Plagues, diseases, weather conditions; all of these things would have affected the health of the animals and the health of people,” Stinson says. “And records of these events might be written down on the skins of the very animals from that region.”

The dozens of documents on which Stinson and team tested their noninvasive collection technique spanned centuries and across several continents. But they all shared one thing in common—being made of parchment.

“We were trying to prove that the technique worked well for parchments from all across Europe, Northern Africa, the Middle East, and across time,” Stinson says.

Thoroughly testing the technique on a wide range of parchment was critical, Stinson explains, because parchment manufacturing methods varied greatly across different regions and eras.

Now, the interdisciplinary research team will apply its groundbreaking technique to a relatively understudied form of parchment known as manor court rolls.

Paper’s predecessor

Before paper, there was parchment. Made from animal skin, Stinson breaks the parchment manufacturing process into three steps: First, soak the skin in water and limestone to help remove all the hair and flesh; then, stretch the skin on a rack until it becomes tight and opaque; and, finally, thin the parchment by carefully shaving and tightening the skin, repeating as many times as necessary to reach the desired thickness.

Parchment is an extremely durable material that can be made as thin as tissue paper. Prior to the advent of the printing press, it was the predominant writing surface—which included the entire medieval era, or about 1,000 years, from the 5th century to the 15th century.

Parchment existed before the 5th century, too, and it’s still used for religious texts by certain cultures to this day. The vast majority of historical parchment samples, though, can be traced to medieval times.

There are roughly a million books made of parchment still intact from the medieval era, Stinson says, and those books, by and large, have received the most attention from historians. But parchment comes in many forms, the vast majority of which are not books. Stinson says that as many as 3 billion pieces of parchment may still survive, scattered somewhere across the globe. And there’s one form of parchment, in particular, that Stinson and his team have now set their sights on.

An untapped resource

In Feudal Europe, large agricultural estates—typically run by nobility and staffed by serfs—were known as manor houses. Manor houses not only kept copious amounts of agricultural records but they also often served as the de facto local court.

“Manor courts would deal with local disputes, but also things like birth, death, change of ownership for land,” Stinson says.

The records manor courts would maintain—be it seasonal crop yields or marriage certifications—were documented on pieces of parchment that came to be known as manor court rolls.

“They would write down all the records of what was decided and when these sessions were held, and when they ran out of space, they would simply sew another skin to the bottom and just keep rolling it up,” Stinson explains.

Eventually, the parchment pieces would often turn into scrolls—some of which can be quite large, Stinson says. That means manor court rolls can cover the happenings of a particular locality over the course of 200 years or more, Stinson says.

Largely thanks to the noninvasive DNA collection method—using cytology brushes—that the team validated last year, they now have the chance to study manor court rolls unlike anyone else has before.

Meiklejohn says manor court rolls present “an untapped resource of biological material.”

“We have a very unique opportunity to learn more about not only the use of animals but also the practices people employed to produce those parchment skins over the centuries,” Meiklejohn says.

After Stinson thoroughly reviews the samples and gives the team the context needed to find relevant clues, Meiklejohn completes the preliminary DNA isolation, high-throughput sequencing and analysis to determine the source species. She then passes the samples along to Breen and Callahan.

Breen, a professor of genomics and the Oscar J. Fletcher Distinguished Professor of Comparative Oncology Genetics, can use digital-droplet PCR to determine the sex of the animal whose skin was used to make the parchment.

Afterward, Callahan, an associate professor of microbiomes and complex microbial communities, can analyze the long-gone animal’s microbiome, which can contain much more genetic information than just that of the target species. In other words, it offers the chance to learn more about the other organisms an animal came in contact with shortly before death.

Over the coming months, Stinson, Meiklejohn, Breen and Callahan will study a collection of manor rolls from three separate sources—the Harvard Law Library, Folger Shakespeare Library and Norfolk Record Office.

Stinson and Meiklejohn believe their team’s research could put NC State at the forefront of an emerging field known as biocodicology—the study of biological clues in manuscripts, books and other documents.

New methods, old problems

While biocodicology remains a nascent field for now, the clues that researchers like Stinson uncover could ultimately help solve problems that have afflicted humanity throughout much of recorded history.

“We’re living in an age of things like COVID, and bird flu, and swine flu,” Stinson says. “And in many ways that’s not new. Some versions of epidemics have been around for a long time.”

You’ve likely heard of the Bubonic plague, colloquially known as the Black Death, but there were plenty of other plagues throughout the medieval era, many of which affected livestock instead of humans, Stinson says. And oftentimes, as was the case with the infamous Bubonic plague of the 1300s, diseases can originate in one species and then spread to others.

Bird flu and swine flu are more recent examples of zoonotic diseases, infectious illnesses that can spread between different animals, including humans. If the end of that last sentence gave you pause, perhaps you’re unfamiliar with the concept of “One Health”—a framework that recognizes the interconnected nature of plant, animal, human and environmental health.

By cataloging human-animal interactions over hundreds of years, manor court rolls provide an unparalleled opportunity to delve deeper into the entwined relationship between human and animal health.

Manor court rolls might have recorded data on some of the earliest known epidemics. And since it’s possible to trace these special parchment samples to a specific time and place—perhaps even the exact farm—they could shed more light on how certain diseases started and progressed.

What’s more, it’s logical to presume that pieces of parchment were made from skins of the same livestock a farmer was keeping records on—which means biological evidence of diseases themselves might still remain.

“Some of the very pathogens that killed those animals may well be on the skins of some of these books,” Stinson says. “So it’s a chance to go back in time and look at the history of epidemics, and how they affected both animals and humans.”

Whether Stinson and Meiklejohn’s interdisciplinary team finds the clues it’s looking for or not, the full story will undoubtedly remain far from finished. But by verifying last year that cytology brushes can collect cellular material without damaging medieval-era documents, they’ve made it much easier for others to help write the next chapter.