Dead Sea Scrolls 'puzzle' pieced together with DNA

The research, conducted over seven years, was published as the cover story in the journal Cell on June 2 and sheds new light on the Dead Sea Scrolls.

"There are many scroll fragments that we don't know how to connect, and if we connect wrong pieces together it can change dramatically the interpretation of any scroll. Assuming that fragments that are made from the same sheep belong to the same scroll," explains Prof. Rechavi, "it is like piecing together parts of a puzzle."

The Dead Sea Scrolls refers to some 25,000 fragments of leather and papyrus discovered beginning in 1947, mostly in the Qumran caves but also in other sites located in the Judean Desert.

Among other things, the scrolls contain the oldest copies of biblical texts. Since their discovery, scholars have faced the breathtaking challenge of classifying the fragments and piecing them together into the remains of some 1,000 manuscripts, which were hidden in the caves before the destruction of the Second Temple in 70 CE.

Researchers have long been puzzled as to the degree this collection of manuscripts, a veritable library from the Qumran caves, reflects the broad cultural milieu of Second Temple Judaism, or whether it should be regarded as the work of a radical sect (identified by most as the Essenes) discovered by chance.

"Imagine that Israel is destroyed to the ground, and only one library survives -- the library of an isolated, 'extremist' sect: What could we deduce, if anything, from this library about greater Israel?" Prof. Rechavi says. "To distinguish between scrolls particular to this sect and other scrolls reflecting a more widespread distribution, we sequenced ancient DNA extracted from the animal skins on which some of the manuscripts were inscribed. But sequencing, decoding and comparing 2,000-year old genomes is very challenging, especially since the manuscripts are extremely fragmented and only minimal samples could be obtained."

Pnina Shor, founder of the Dead Sea Scrolls Unit at the Israel Antiquities Authority, adds, "The Israel Antiquities Authority is in charge of both preserving the scrolls for posterity and making them accessible to the public and to scholars. Recent scientific and technological advances enable us to minimize physical intervention on the scrolls, thus facilitating multidisciplinary collaborations."

Innovative methods to solve historical mysteries

To tackle their daunting task, the researchers developed sophisticated methods to deduce information from tiny amounts of ancient DNA, carefully filtering out potential contaminations and statistically validating the findings. The team employed these mechanisms to deal with the challenge posed by the fact that genomes of individual animals of the same species (for instance, two sheep of the same herd) are almost identical to one another, and even genomes of different species (such as sheep and goats) are very similar.

For the purpose of the research, the Dead Sea Scrolls Unit of the Israel Antiquities Authority supplied samples -- sometimes only scroll "dust" carefully removed from the uninscribed back of the fragments -- and sent them for analysis by Prof. Rechavi's team: Dr. Sarit Anava, Moran Neuhof, Dr. Hila Gingold and Or Sagi. To prevent DNA contamination, Dr. Anava traveled to Sweden to extract the DNA under the meticulous conditions required for ancient DNA analysis (e.g. wearing special full-body suits) in Prof. Jakobsson's paleogenomics lab in Uppsala, which is equipped with cutting-edge equipment. In parallel to the teams that were studying the animals' ancient DNA, Prof. Mason's metagenomics lab in New York studied the scrolls' microbial contaminants. Prof. Jakobsson says, "It is remarkable that we were able to retrieve enough authentic ancient DNA from some of these 2,000 year old fragments considering the tough history of the animal hides. They were processed into parchment, used in a rough environment, left for two millennia, and then finally handled by humans again when they were rediscovered."

Textual pluralism opens window into culture of Second Temple Jewish society

According to Prof. Rechavi, one of the most significant findings was the identification of two very distinct Jeremiah fragments.

"Almost all the scrolls we sampled were found to be made of sheepskin, so most of the effort was invested in the very challenging task of trying to piece together fragments made from the skin of particular sheep, and to separate these from fragments written on skins of different sheep that also share an almost identical genome," says Prof. Rechavi. "However, two samples were discovered to be made of cowhide, and these happen to belong to two different fragments taken from the Book of Jeremiah. In the past, one of the cow skin-made fragments was thought to belong to the same scroll as another fragment that we found to be made of sheepskin. The mismatch now officially disproves this theory.

"What's more, cow husbandry requires grass and water, so it is very likely that cow hide was not processed in the desert but was brought to the Qumran caves from another place. This finding bears crucial significance, because the cowhide fragments came from two different copies of the Book of Jeremiah, reflecting different versions of the book, which stray from the biblical text as we know it today."

Prof. Mizrahi further explains, "Since late antiquity, there has been almost complete uniformity of the biblical text. A Torah scroll in a synagogue in Kiev would be virtually identical to one in Sydney, down to the letter. By contrast, in Qumran we find in the very same cave different versions of the same book. But, in each case, one must ask: Is the textual 'pluriformity,' as we call it, yet another peculiar characteristic of the sectarian group whose writings were found in the Qumran caves? Or does it reflect a broader feature, shared by the rest of Jewish society of the period? The ancient DNA proves that two copies of Jeremiah, textually different from each other, were brought from outside the Judean Desert. This fact suggests that the concept of scriptural authority -- emanating from the perception of biblical texts as a record of the Divine Word -- was different in this period from that which dominated after the destruction of the Second Temple. In the formative age of classical Judaism and nascent Christianity, the polemic between Jewish sects and movements was focused on the 'correct' interpretation of the text, not its wording or exact linguistic form."

Identification of genetically distinct groups of sheep suggests prominence of ancient Jewish mysticism

Another surprising finding relates to a non-biblical text, unknown to the world before the discovery of the Dead Sea Scrolls, a liturgical composition known as the Songs of the Sabbath Sacrifice, found in multiple copies in the Qumran caves and in Masada. Apparently, there is surprising similarity between this work and the literature of ancient Jewish mystics of Late Antiquity and the Middle Ages. Both Songs and the mystical literature greatly expand on the visionary experience of the divine chariot-throne, developing the vision of the biblical prophet Ezekiel. But the Songs predates the later Jewish mystical literature by several centuries, and scholars have long debated whether the authors of the mystical literature were familiar with Songs.

"The Songs of the Sabbath Sacrifice were probably a 'best-seller' in terms of the ancient world," Prof. Mizrahi says. "The Dead Sea Scrolls contain 10 copies, which is more than the number of copies of some of the biblical books that were discovered. But again, one has to ask: Was the composition known only to the sectarian group whose writings were found in the Qumran caves, or was it well known outside those caves? Even after the Masada fragment was discovered, some scholars argued that it originated with refugees who fled to Masada from Qumran, carrying with them one of their scrolls. But the genetic analysis proves that the Masada fragment was written on the skin of different sheep 'haplogroup' than those used for scroll-making in Qumran. The most reasonable interpretation of this fact is that the Masada Scroll did not originate in the Qumran caves but was rather brought from another place. As such, it corroborates the possibility that the mystical tradition underlying the Songs continued to be transmitted in hidden channels even after the destruction of the Second Temple and through the Middle Ages."

From solved riddles to new mysteries: Yet undiscovered caves?

Since most of the scrolls were found to be written on sheepskin, the team had to find a way to distinguish "in higher resolution" between the very similar genomes of different sheep.

"Mitochondrial DNA can tell us whether it is a sheep or a cow, but it can't distinguish between individual sheep," Prof. Rechavi adds. "We developed new experimental and informatic methods to examine the bits of preserved nuclear DNA, which disintegrated over two millennia in arid caves, and were contaminated in the course of 2,000 years, including recently by the people who handled the scrolls, often without even the use of gloves."

Using these methods, it was discovered that all the sampled scroll-fragments written using a particular scribal system characteristic to the sectarian writings found in the Qumran caves (the "Qumran scribal practice") are genetically linked and differ collectively from other scroll-fragments that were written in different ways and discovered in the very same caves. This finding affords a new and powerful tool for distinguishing between scrolls peculiar to the sect and scrolls that were brought from elsewhere, and potentially reflect the broader Jewish society of the period.

Shor says, "Such an interdisciplinary project is very important indeed. It advances Dead Sea Scrolls research into the 21st century, and may answer questions that scholars have been debating with for decades. We consider the present project, which integrates both extraction of genetic information from the scrolls using novel methods together with classical philological analysis, a very significant contribution to the study of the scrolls."

The project examines not only scroll fragments but also other leather artifacts discovered at various sites throughout the Judean Desert. The genetic differences between them have allowed researchers to discern between different groups of findings.

According to Prof. Mizrahi, many scroll fragments were not found by archaeologists, but by shepherds, delivered to antiquity dealers, and only subsequently handed over to scholars.

"We don't always know precisely where each fragment was discovered, and sometimes false information was given about this matter," says Prof. Mizrahi. "Identifying the place of discovery is important, because it affects our understanding of the historical context of the findings. For this reason, we were excited to learn that one fragment, that was suspected to originate not from Qumran but rather from another site, indeed had a 'genetic signature' that was different from all the other scrolls found in the Qumran caves sampled for this research."

But this finding led to yet another enigmatic discovery pertaining to a fragment containing a text from the Book of Isaiah. This fragment was published as a Qumran scroll, but its genetic signature also turned out to be different from other scrolls in Qumran.

Prof. Mizrahi concludes, "This raises a new curious question: Was this fragment really found in the Qumran caves? Or was it originally found in yet another, unidentified location? This is the nature of scientific research: We solve old puzzles, but then discover new mysteries."