by Pbo, Svante
One bright spot, late in the summer of 2006, was the arrival of a talented Croatian graduate student, Tomislav Maričić, in our group. Tomi had accompanied us when we visited the Institute for Quaternary Paleontology and Geology, and his cultural connections with Croatia came in handy as we tried to reach an agreement over the Croatian Neanderthals. Our project had become a matter of public debate there—a debate I could follow thanks to Tomi’s translations of the Croatian newspapers. In July, after we had announced the Neanderthal Genome Project at the press conference in Leipzig, one of the big dailies, Jutarnji List, interviewed Jakov Radovčić, who was described as someone “without whom no Neanderthal research can be imagined.” Said Jakov: “The question is: What is the goal of the research? Also, it is still not clear that one can retrieve the whole Neanderthal genome. . . . They are using a chemically aggressive method which destroys the material, which is too precious for us to sacrifice.” In November the same paper quoted him again: “Three and a half months ago, Svante Pääbo was in Zagreb looking for more samples for his molecular genetic analysis. . . . However, I think we should take special care of the samples and keep them safe, so the next generation of researchers can use them.”
Prompted by these remarks, I sent Jakov a long, polite e-mail, in which I once again explained our project. He answered that after some curatorial formalities that were likely to take “several weeks or a few months,” he would “strongly support” our project. Meanwhile, rumors were flying hither and yon in Zagreb. It was frustratingly unclear to me just who supported and who was against the project, what was said by whom, and whether people really meant what they said to me directly. The only people in whom I had solid confidence were Pavao Rudan and two friends of his, both members of the Croatian Academy of Sciences and Arts, who supported us. One of these was Željko Kučan, a statesman-like scientist of poise and judgment who had been the first to introduce the study of DNA at Zagreb University, some fifty years earlier. The other was a geologist named Ivan Gušić, known as “Johnny” to his friends. Jovial, positive, and always friendly, Johnny was soon to become the new head of the Institute for Quaternary Paleontology and Geology (see Figure 12.2).
In late November, Pavao used the occasion of the publication of our Nature and Science papers to take a public stand in our favor. He wrote an article about the project in the Sunday edition of Vjesnik, the Croatian newspaper of record, emphasizing that DNA studies could reveal much about human evolution and that the Vindija material was essential for this. “Therefore, the cooperation with the Max Planck colleagues should be continued and made stronger,” he argued. “It is the Vindija samples that are kept in a HAZU [the Croatian Academy’s acronym] collection that can make it possible to retrieve a Pleistocene hominid genome for the first time in history. . . . Future cooperation between HAZU and [the] Berlin-Brandenburg Academy, especially with Svante Pääbo’s team, will improve paleoanthropological, molecular genetics, and anthropological science.” I very much hoped that our work would eventually show that Pavao had not misplaced his trust.
Slowly the Croatian tide turned in our favor. On December 8, 2006, after many vicissitudes, most of them incomprehensible, a memorandum of understanding between the Zagreb and Berlin academies was signed. What a relief! Finally nothing stood between us and the bones. As soon as was feasible, I arranged to visit Zagreb with Johannes and Christine Verna, a young French paleontologist from the Department of Human Evolution in our institute in Leipzig. She was to spend ten days at the Institute for Quaternary Paleontology and Geology making a preliminary catalog of all the Neanderthal bones in the Vindija collection. Johannes and I spent four days in Zagreb and then returned to Leipzig in the company of Pavao, Željko, and Johnny, who carried eight bones from Vindija in sterile bags, including the celebrated Vi-80, now officially known as Vi-33.16 (see Fig. 12.1).
We arrived late at night. The first thing we did next morning was to bring the bones to the Department of Human Evolution, where, still in their bags, they were scanned by computer tomography so that their morphology would be forever preserved in a digital form. Then the bones went into the clean room, and Johannes took over.
Using a dental drill with a sterilized bit, he removed two or three square millimeters of the surface from each bone. Then he drilled a small hole into the compact part of each bone, pausing frequently to avoid heating the bone and potentially damaging the DNA (see Figure 12.3). He collected about 0.2 grams of bone, adding it to a solution that within a few hours bound the bone’s calcium. What was then left of the bone was a pellet of proteins and other components from its nonmineral portion. The DNA, however, was in the dissolved liquid part, and Johannes purified it by letting it bind to silica—the technique that Matthias Höss, fourteen years earlier, had found to be particularly good at isolating DNA from ancient bones.
Figure 12.2. Pavao Rudan, Željko Kučan, and Ivan “Johnny” Gušić, the three members of the Croatian Academy of Sciences who made it possible for us to sample the Neanderthal bones from Vindija Cave. Photo: P. Rudan, HAZU.
To make the DNA molecules amenable to 454 sequencing, Johannes used enzymes to fill in and chew away any unraveled single-stranded DNA at the ends of molecules. That enabled him to use a second enzyme to fuse short synthetic pieces of modern DNA, called adaptors, to the ends of the ancient DNA. After adaptors have been added to DNA molecules, they can be “read” by sequencing machines just like books, so the collection of them is called a library. The adaptors had been synthesized especially for this project, and they contained a short additional sequence of four bases, TGAC, positioned so that it would abut the ancient fragments as a kind of marker or tag. This was one of those small technical details that often make a huge difference in molecular biology in general and ancient DNA research in particular. We had introduced these tags because our ancient DNA library had to leave the clean room to be sequenced on the 454 machine. In order to ensure that DNA from other libraries in our lab could not somehow end up in the Neanderthal libraries, we used these special adaptors and trusted only sequences that started with TGAC. We described this adaptor innovation in a 2007 paper.{51}
Using these procedures, Johannes prepared extracts and libraries from the eight new Vindija bones. He then used the PCR to see if there was Neanderthal mtDNA in the extracts and to estimate the extent of modern human contamination. Nearly all of the bones contained Neanderthal mtDNA. This was encouraging, but after our disappointments with the bones from Russia, Germany, and Spain, I did not allow myself to get enthusiastic. We immediately sequenced a sample of random DNA fragments from each of the libraries to estimate their proportion of nuclear Neanderthal DNA, and for the few days it took to get the results, I could hardly concentrate on any of my other work. We had announced to the world that we would sequence the Neanderthal genome. If these new Vindija bones did not contain enough nuclear DNA to do so, I was certain that we would have to announce our failure. I did not know where to look for any better bones.
Figure 12.3. Sampling a Neanderthal bone with a sterile drill. Photo: MPI-EVA.
When the results were in, they showed that some of the bones contained between 0.06 and 0.2 percent Neanderthal nuclear DNA, similar to what we had seen at the other sites. But three bones contained more than 1 percent Neanderthal nuclear DNA, and one contained nearly 3 percent. This was our favorite bone Vi-33.16, aka Vi-80. We had not found the magical bone with huge amounts of nuclear Neanderthal DNA that we had hoped for, but it was a bone we could work with.
All was not lost.
Chapter 13
The Devil in the Details
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Over the Christmas and New Year holidays I used my free time to ponder our situation. It was sobering. When I calculated how much of such bones we would need to complete the genome sequencing I came to tens of grams, more than the entire weight of the bones we had. I felt very bad. Had I been hopelessly overoptimistic, or naïve, to think that we could do it? Had I been fool
hardy to think that we would find a bone from Vindija Cave that contained more Neanderthal DNA than the first ones we had analyzed? Had I put too much belief in 454 magically coming up with more powerful sequencing machines that would somehow allow us to sequence more? Why had I risked my calm and orderly scientific life on this gamble, which it now seemed I was more than likely to lose?
My twenty-five years in molecular biology had essentially been a continuous technical revolution. I had seen DNA sequencing machines come on the market that rendered into an overnight task the toils that took me days and weeks as a graduate student. I had seen cumbersome cloning of DNA in bacteria be replaced by the PCR, which in hours achieved what had earlier taken weeks or months to do. Perhaps that was what had led me to think that within a year or two we would be able to sequence three thousand times more DNA than what we had presented in the proof-of-principle paper in Nature. Then again, why wouldn’t the technological revolution continue? I had learned over the years that unless a person was very, very smart, breakthroughs were best sought when coupled to big improvements in technologies. But that didn’t mean we were simply prisoners awaiting rescue by the next technical revolution. Perhaps, I thought, we could help the technology along a bit.
I reasoned that since we had so little bone, and since it contained so little DNA, we needed to minimize losses of DNA from extraction to library. At our first Friday meeting after the holiday breaks I tried to instill a sense of acute crisis in the group. I said that it was now clear that we were not going to find a magical bone that would save us by containing lots of Neanderthal DNA. We needed to make do with what we had, and that meant rethinking every single step we did in the lab. I argued that the losses were probably huge. For example, the procedures used to purify DNA produced solutions that contained only minute quantities of other components, such as proteins. But the price of such purity is the loss of much DNA. If we could minimize such losses, perhaps the bones we had would be enough—at least once 454 Life Sciences finally had its new, more efficient machines ready.
I cross-examined my group week after week, asking repeatedly about every step they did in the lab. Perhaps the strategy of returning to the same questions over and over again was something I had retained from my training as an interrogator of prisoners of war during my otherwise long-forgotten military training in Sweden in my youth. The more I asked, the more I came to suspect that the recommended 454 protocols for preparing sequencing libraries, which were heavy on purification, might lead to undue loss of DNA. I insisted that we systematically analyze each step. How could we best do that?
When I was a graduate student the use of radioactivity was central to almost every experiment in molecular biology, but the cumbersome safety precautions required had long since inspired biologists to use nonradioactive assays. As a consequence, biology students today have almost no experience in working with radioactivity. However, radioactive labeling remains one of the most sensitive ways to detect tiny amounts of DNA. So in one of our Friday meetings, I suggested that Tomi Maricic label a small amount of DNA with radioactive phosphorus and use it to prepare a sequencing library. He could then collect the side fractions that were normally discarded and measure how radioactive they were. The amount of radioactivity he detected in a side fraction would directly measure the loss of DNA at that step.
I assumed that the silence which greeted this idea in our Friday meeting was a tribute to the quiet elegance of this approach. But really I had run headlong up against an aspect of how our group functions. This aspect is, I believe, one of its biggest strengths, but at times it proves a weakness. I have encouraged a culture in which all ideas are discussed; everyone in the meeting is expected to speak his or her mind, and in the end we try to reach a consensus about what should be done. But as in any democracy, irrational ideas can sometimes win the day. My radioactivity plan aroused skepticism in several people who were influential in the group. They made a number of objections to it, fueled (I thought) by an unconscious reluctance to adopt a method they had little experience with and which sounded old-fashioned and unsafe, if not downright scary. I decided not to force the issue. Other methods were tried instead, such as measuring the DNA amounts in each step of the library preparation and using more modern PCR-based approaches. But these methods either were not sensitive enough or proved ineffective in other ways. Over the next few months, I continued to suggest the radioactivity experiment, with increasing impatience, at times longing for a return to a more autocratic era, when the professor’s word was law. Yet still I acquiesced, not wanting to put a chill on the free-floating exchange of ideas I felt was so valuable in the group.
Finally, when all other efforts had come to naught, it was the group that acquiesced. Tomi reluctantly ordered some radioactive phosphorus, labeled some ordinary human DNA we used for test purposes, and took it through the steps of preparing a 454 sequencing library. The results were stunning. He showed that in each of the first three major steps in the preparation, between 15 and 60 percent of the DNA was lost—a level not entirely unexpected in a biochemical separation. But in the last step, where the complementary DNA strands were separated with a strong alkaline solution, more than 95 percent of the input DNA was lost! Others who used this separation method with ordinary modern DNA had not noticed its inefficiency, because they had so much DNA that these enormous losses didn’t matter to them. For our ancient work, though, they were catastrophic. Once the problem was identified, a simple remedy was devised. Alkaline solutions are not the only way to separate DNA strands; they also separate when they are heated. So Tomi tried heating and found from 10 to 250 times more radioactivity in the final DNA preparation! This was a great, indeed game-changing, advance.
Most labs discard side fractions as by-products. Fortunately, we had saved all of ours from our previous experiments. For years I had insisted on doing so, just in case something came along that would make them useful. This was easily one of my least popular ideas and caused many freezers to be filled with frozen side fractions that no one thought would ever be used. But thankfully in this case the crazy idea of the professor had been adhered to by the group. So now Tomi could simply heat the side fractions from earlier library preparations from the Vindija bones and retrieve additional, relatively copious amounts of Neanderthal DNA without even having to do any more extractions. He also optimized other steps in the library preparation. These changes resulted in a protocol several hundred times more efficient in turning the extracted DNA into a library ready for sequencing.{52}
Following consultation with our Croatian partners, we dedicated three Vindija bones—Vi-33.16 along with two new bones, Vi-33.25 and Vi-33.26—to the project. All seemed to be fragments of long bones that had apparently been crushed to get at the marrow (see Figure 12.1). Thanks to Tomi’s advance we could now in principle produce libraries that contained 3 billion nucleotides of Neanderthal DNA from just these three bones. But the libraries would still contain at least 97 percent bacterial DNA, so the people in Branford would need to do between four thousand and six thousand runs on their sequencing machines to arrive at 3 billion base pairs of Neanderthal DNA. This was far more than we could ever imagine convincing Michael Egholm to do.
It seemed to me we were still stuck, until someone suggested that perhaps we could find pockets in our three bones where they contained much less bacterial DNA and therefore, relatively speaking, more Neanderthal DNA. Now and again over the years we had indeed seen indications that some parts of a bone might contain higher amounts of bacterial DNA than others, perhaps because bacteria had found growth conditions better in one part of the bone, and therefore multiplied more there, than in other parts. So, fueled by this hope, Johannes tried to systematically identify the best regions to sample. He drilled the bones until they looked first like flutes and then like Swiss cheese. He did indeed find a 10-fold difference in the percentage of Neanderthal DNA in regions just a centimeter or two apart, but the best regions still contained no more than 4 percent Neander
thal DNA!
We came back to this problem again and again in our Friday meetings. To me, these meetings were absorbing social and intellectual experiences: graduate students and postdocs know that their careers depend on the results they achieve and the papers they publish, so there is always a certain amount of jockeying for opportunity to do the key experiments and to avoid doing those that may serve the group’s aim but will probably not result in prominent authorship on an important publication. I had become used to the idea that budding scientists were largely driven by self-interest, and I recognized that my function was to strike a balance between what was good for someone’s career and what was necessary for a project, weighing individual abilities in this regard. As the Neanderthal crisis loomed over the group, however, I was amazed to see how readily the self-centered dynamic gave way to a more group-centered one. The group was functioning as a unit, with everyone eagerly volunteering for thankless and laborious chores that would advance the project regardless of whether such chores would bring any personal glory. There was a strong sense of common purpose in what all felt was a historic endeavor. I felt we had the perfect team (see Figure 13.1). In my more sentimental moments, I felt a love for each and every person around the table. This made the feeling that we’d achieved no progress all the more bitter.
