Neanderthal Man

by Pbo, Svante

  But we were not the only ones thinking of trying pyrosequencing on ancient DNA. Early in 2006, while Ed Green was busy analyzing our cave-bear and mammoth data, a paper appeared in Science by my previous graduate student, Hendrik Poinar, now at McMaster University in Ontario, in collaboration with Stephan Schuster at Penn State University. They used pyrosequencing applied directly to the DNA extract, just as we had done with 454 Life Sciences, to determine 28 million nucleotides of DNA from a permafrost mammoth.{46} I was happy that a previous student was doing this, even if our group was disappointed not to be the first to publish sequencing of ancient DNA using pyrosequencing. We had had our data from the mammoth and cave-bear bones for many months, but we’d spent a lot of time on two things the Science paper did not: analyzing how best to match the DNA sequences we had determined to reference genomes and considering how errors in the sequences would affect the results. Still, Hendrik’s paper was more evidence that direct sequencing was the way to go. It also showed, once again, that permafrost material could be amazingly well preserved. About half of the DNA in Hendrik’s sample was from the mammoth, a far cry from what we could hope for from our Neanderthals—we were happy when we had an extract containing 1 or 2 percent Neanderthal DNA. Hendrik’s paper also illustrated a dilemma in science: doing all the analyses and experiments necessary to tell the complete story leaves you vulnerable to being beaten to press by those willing to publish a less complete story that nevertheless makes the major point you wanted to make. Even when you publish a better paper, you are seen as mopping up the details after someone who made the real breakthrough. Our group discussed this point extensively after Hendrik’s paper appeared. Some felt we should have published earlier. In the end, the analysis of our cave-bear and mammoth sequences appeared in the September 2006 issue of Proceedings, where we, ironically, ended up reporting erroneous conclusions about deaminated A’s giving rise to mutations in the sequences.{47}

  In May each year, there is a meeting on genome biology at Cold Spring Harbor Laboratory on Long Island. This meeting is the unofficial summit of genome scientists from around the world, and presenters are expected to talk about novel and unpublished results. It tends to be an intense affair, colored by the rivalry among genome centers and sometimes by conflicts and aggressions carried over from the race to sequence the human genome.

  In 2006, the genome meeting was even more intense for me than it normally is. We had just obtained Neanderthal sequencing results both from 454 Life Sciences and from Eddy Rubin’s group at Berkeley, and we had done some preliminary analyses. I had two goals for my talk. First, I wanted to present the comparisons of the two different techniques to sequence ancient DNA. Second, I wanted to lay out a road map of how one might get to a whole-genome sequence of the Neanderthal and other extinct organisms. The results confirmed that direct pyrosequencing was the method of the future, so my emphasis would be on that.

  I was unusually nervous when I arrived at Cold Spring Harbor. I was housed in a small spartan room on campus, an honor bestowed on frequent attendees of the meeting, while others have to be bused in from far-flung hotels. I spent the entire flight to New York, as well as the first night in my tiny room, preparing my talk. The next day I collected the people from my lab who attended the meeting and I gave a practice talk in a side hallway. I had the feeling that this would be a talk that would define what we were to do in the next few years.

  It is rare to have the undivided attention of the audience when giving a scientific talk. The Genome Biology Meeting at Cold Spring Harbor is a case in point. I had given many talks there before and was used to watching most of the six hundred or so people in the room fiddle with their laptops as they checked through their own presentations or e-mailed colleagues—or dozed off due to the combined effects of jet lag and too many highly detailed talks. But this time was different. As I worked my way through the mammoth and cave-bear results toward the Neanderthal data, I could feel how I had the absolute and undivided attention of the audience. My last slide was a map of the human chromosomes, with little arrows showing where the tens of thousands of pieces of DNA we had sequenced from the Neanderthal matched. When the slide went up, I heard what sounded like a gasp from the audience. Our sequences added up to only about 0.0003 percent of the Neanderthal genome, but it was clear to everyone that we had shown that one could now—in principle—sequence the whole thing.

  Chapter 11

  Starting the Genome Project

  ________________________________________

  That night, after returning to my little room at Cold Spring Harbor Laboratory, I lay down on the bed and stared at the ceiling. So far, I had had a nice—one might even say somewhat distinguished—career. I had a permanent research position with solid funding, was doing interesting projects, and got invited several times a year to give talks around the world. Now I had really stuck my neck out, publicly promising to sequence the Neanderthal genome. If we succeeded, it would clearly be my biggest achievement to date; but if we failed, it would be a very public embarrassment, almost surely a career-ending one. And I knew that succeeding would not be as easy as I had made it sound in my talk. We needed three things to succeed: many 454 sequencing machines, lots more money, and good Neanderthal bones. We had none of them, but fortunately no one else seemed to realize this. I knew it only too well, however, and I lay in bed a long time, with all the things we needed to make the project possible running through my head.

  The first priority was access to lots of sequencing machines from 454 Life Sciences. An obvious move would be to visit Jonathan Rothberg in Branford, Connecticut, which was not far from Cold Spring Harbor. Next morning at breakfast, I collected the key people involved in our Neanderthal work, all of whom were at the meeting: Ed Green, Adrian Briggs, and Johannes Krause. After breakfast, we jumped into my rental car and took off for Branford. I have a deplorable tendency to pack too many commitments into too little time and as a consequence am chronically late for appointments, flights, and other scheduled activities. This outing was no exception. As we drove toward Port Jefferson on northern Long Island, we realized we were probably going to miss the ferry across the Long Island Sound to Bridgeport. As it happened, we were the last car to squeeze aboard (in fact, the rear of the car jutted out over the water as we steamed across). I hoped this close call was a good omen.

  This was the first of what would be several visits to 454 Life Sciences. Jonathan Rothberg was just as intense and full of maverick ideas in person as he had been on the phone. For balance, there was Michael Egholm, the practical-minded Dane concerned with reality checks and getting things done. As the project progressed, I came to appreciate both men immensely; between Jonathan’s vision and drive and Michael’s down-to-earth practicality, they made a terrific pair. Our discussion that day was dominated by what it would take to sequence the Neanderthal genome. It was clear that we would apply the “shot-gun” technique that Craig Venter had introduced and used in his bid to sequence the human genome at his company Celera. This approach involved sequencing random fragments and then putting them together computationally by looking for overlaps between fragments. One major complication involves repetitive DNA sequences in the genome; such sequences make up about half of the genomes of humans and apes. Most of these repetitive sequences are a few hundred or even thousands of nucleotides long, and many occur not only a few but many thousands of times across the genome. Therefore, shot-gun approaches typically use not only short DNA fragments but also longer fragments so that one can “bridge” such repeat sequences with fragments that “anchor” it in single-­copy sequences on each side of the repeat. This makes it possible to know where each repeat element sits in the genome. But our ancient DNA was already broken down into short pieces. Therefore, we planned to use the human reference genome (the first human genome, sequenced by the public genome project) as a template for reconstructing the Neanderthal sequences. But while this should work for DNA sequences that occurred a single time in the genome, we could
not hope to determine the sequences of all the repetitive parts. To me, it seemed a small sacrifice: the single-copy sequences tend to be the most interesting parts of a genome as they contain the most genes with well-known functions.

  We also needed to decide how much of the genome to sequence. Before visiting 454 Life Sciences, I had decided to sequence about 3 billion nucleotides from our Neanderthal bones. This goal was dictated mostly by what I thought was possible, and also because it was approximately the size of the human genome. The fragmented nature of the ancient DNA meant that we would get sequences of many bits of the genome just once; other bits twice, from two independent fragments; others three times; and so on. It also meant that there were many parts of the genome we would not see at all, simply because no DNA fragment we sequenced would happen to include them. Statistically, we could expect to get two-thirds of the entire genome at least once and so fail to see about one-third. In genome-speak, this is known as 1-fold coverage, since statistically each nucleotide has the chance to be seen once. I felt that 1-fold coverage was a feasible goal, and one that would provide a good overview of the Neanderthal genome. Importantly, the resulting genome would be a stepping stone of sorts. Future sequences, derived from other Neanderthals, could be put together with ours to arrive at higher “coverage” until eventually all of the genome, at least the parts that were not repetitive, had been seen.

  The goal I had set ourselves was thus somewhat arbitrary. Compared with sequencing efforts expended on present-day genomes, it was also rather humble, as those other projects aimed for 20-fold coverage or more. However, the task was still monumental. Our very best extracts contained just 4 percent Neanderthal DNA. I counted on finding more such bones and hoped that some would contain even a bit more Neanderthal DNA, assuming that if the average stayed at 4 percent, to get our 3 billion nucleotides we would have to generate some 75 billion nucleotides in all. And since our fragments were short, 40 to 60 nucleotides on average, this added up to between 3,000 and 4,000 runs on the new sequencing machines. It was the equivalent of devoting the entire facility at 454 Life Sciences to the Neanderthal project for many months—and at normal customer prices, it would be impossible for us even to contemplate.

  Ed, Adrian, Johannes, and I discussed all this with Jonathan and Michael. The project clearly had appeal not only to Jonathan but to 454 Life Sciences as a company, because of its potential both to provide truly unique insights into human evolution and also, more pragmatically, to give 454’s technology even higher visibility. I gladly agreed that the company people would be real scientific partners as well as co-authors on future publications with us, but that didn’t mean we could do sequencing for free. Finally, we arrived at a price: $5 million. I could not decide whether that was good or bad news. It was more money than I had hoped to pay, but not a completely outlandish sum. We said we would go back home and think about it.

  After the negotiations were done, Jonathan offered the four of us take-out sandwiches and sodas and then asked if we wanted to see his house before we headed back to the meeting at Cold Spring Harbor. We agreed. After our late lunch, we followed him home. I had grown up in humble circumstances, and my mother, a refugee from the Soviet invasion of Estonia at the end of World War II, had transmitted a highly pragmatic outlook to me. As a result, I am not easily impressed by luxury. But the visit to Jonathan’s place turned out to be very memorable, even though we never got to see his house. Instead, we visited the grounds where he lived on a peninsula in Long Island Sound. On the beach, he had built an exact replica of Stonehenge—exact, that is, except that it was made of Norwegian granite and therefore heavier than the original, and it was slightly modified to account for how the sun would fall between the stones on the birthdays of his family members. As we walked among the huge monoliths, Jonathan turned to me and said, “Now you probably think I’m crazy.” I of course said no, but not only out of politeness. I really didn’t think Jonathan was crazy. He was deeply fascinated by ancient history, and more important, he had big ideas and was able to turn his dreams into reality. His Connecticut Stonehenge was, I thought, another good omen for our undertaking.

  The next day, back at Cold Spring Harbor, I could not concentrate at all. Five million dollars was a lot of money, about ten times as much as a big research grant in Germany. The Max Planck Society provides generous funding to its institute directors so that they can concentrate on research and not on grant writing, but $5 million was still a much higher amount than the entire yearly budget for my department. I worried that we would need to turn this project over to some genome center, just because we didn’t have the money. Then I remembered Herbert Jäckle, the developmental biologist who had helped persuade me to move to Germany in 1989 when he was professor of genetics in Munich. He, too, had moved to a Max Planck institute—the Institute for Biophysical Chemistry in Göttingen—and had again played an important yet unofficial role in getting me to move, in 1997, from Munich to Leipzig to join in establishing the Institute for Evolutionary Anthropology. In fact, ever since I had come to Germany, when I had faced crucial turning points in my scientific life, Herbert had always been there with support and advice. Now he was vice president of the biomedical section of the Max Planck Society. Fortunately, the society is a research organization where scientists, such as Herbert, rather than administrators or politicians are in charge. That very afternoon I decided to call him from Cold Spring Harbor.

  I don’t call Herbert often, so I think he realized that this was a matter of some import and urgency. When I got him on the line, I described our calculations on the feasibility of sequencing the Neanderthal genome and the cost, and I asked if he had any advice on how one might raise that much money in Europe. He said he would think about it and get back to me in a few days. I flew back to Leipzig the next day, torn between hope and despair. Perhaps we could find a rich benefactor, but how do you find one of those?

  Two days after my return, true to his word, Herbert called me. The Max Planck Society, he said, had recently set up a Presidential Innovation Fund to support extraordinary research projects. He had discussed our project with the society’s president, and the society was ready—in principle—to support us with the requested funds, distributed over three years. They had even reserved the money, pending a written proposal, which would need to be reviewed by experts in the field. I was totally taken aback; I don’t recall even thanking him properly before hanging up. This money made all the difference in the world! I ran from my office into the lab and babbled the news to the first people I met. Then I immediately sat down and started drafting a proposal describing the results and calculations that had convinced us we could sequence the Neanderthal genome within three years, given sufficient resources.

  At the proposal’s conclusion, I had to present a financial plan. When I started working it up, something extremely embarrassing dawned on me. I had called Herbert from the United States and said we needed “5 million” for the project, thinking in terms of US dollars. Herbert, in Europe, must have thought I meant 5 million euros. He may even have said that the Max Planck Society had reserved “5 million euros” for our project, but I had been too excited to register it. At the exchange rate, then, that amounted to US$6 million. What to do? Perhaps I could quietly increase the budget to accommodate the additional 20 percent in funds—but that would be disingenuous and might even be noticed once we signed a contract with 454 Life Sciences. I called Herbert and, with considerable embarrassment, explained the situation. He laughed. Then he asked whether we might not have extra costs in Leipzig, above what we were to pay 454 Life Sciences for sequencing. Of course we would. We would have to extract DNA from many fossils to find good ones, and test them all by making sequencing runs from them ourselves. So we needed to buy our own sequencing machine from 454 for testing all the extracts, and we needed reagents to run it. With the difference the exchange rate made, we could really make the project fly. I was elated and wrote a plan that included the work that would be done at our in
stitute in Leipzig.

  Meanwhile, Eddy Rubin’s group in Berkeley had made a bacterial library of the entire Neanderthal extract we had sent them. Jim Noonan, Eddy’s postdoc, had sequenced every drop. What they had retrieved amounted to a bit over sixty-five thousand base pairs. In Branford, they had used about 7 percent of the extract we had sent them and produced about a million base pairs. So, just as Adrian had predicted, the direct-sequencing approach was about two hundred times more efficient in generating DNA sequences from an extract. Eddy insisted nonetheless that his method could be more efficient, and that we should continue to send extracts to him. This was a fundamental disagreement. I realized with some disquiet that I could no longer in good conscience send extracts to Berkeley, when we could generate so much more data from each extract in Branford. But I put the decision off, thinking that it would become obvious to Eddy that the bacterial cloning was inefficient once we wrote up a manuscript describing the results of the two different approaches.