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The proof-of-principle papers published in 2006 were a huge advance, made possible through high-throughput sequencing technology developed by 454 Life Sciences. Their sequencing machine can analyze millions of strands of DNA all at once, in a process that uncouples the double-stranded DNA, chops it up into fragments, attaches those fragments to minute beads, and creates millions of clones of the original fragments. These beads are then packed into individual wells on a slide, and analyzed in a machine that determines the fragments’ sequences by recording the identity of every base as it binds with its complementary nucleotide.

The new results, Egholm says, relied both on the 454 machine and on one made by Solexa, which can analyze hundreds of millions of wells at once. Speed was key: since only 4 percent of what was sequenced actually belonged to a Neanderthal, the researchers had to sequence over 100 million base pairs of unrelated DNA just to get 3 million base pairs of Neanderthal DNA. “With lower throughput sequencers, it would have been really difficult to generate enough data to sequence the entire genome,” says Rachel Mackelprang, a postdoc in genetic analysis at the Department of Energy Joint Genome Institute.

Using previously sequenced genomes from other species was also crucial, says John Hawks, a biological anthropologist at the University of Wisconsin. “Bootstrapping computer information about genomes really made all of this possible,” he says. “To be able to take snippets of DNA of 50 base pairs or less and have the computer say that it’s the same as a bacterial sequence has enabled the reconstruction of genuine Neanderthal sequence.”

But more than anything, the largest challenge has been finding ways to detect and eliminate sequences from human DNA from the Neanderthal samples. The 2006 research was scrutinized heavily by Pääbo and Egholm’s peers. Despite the researchers’ caution, human DNA had infiltrated the samples and been included in the original sequence. This time around, to correct for contamination, they used a number of technological innovations, including the placement of genetic tags on all bone-derived DNA, which allowed them to detect and discard all untagged DNA as contaminants.

“Having a genome allows us to look for things that we can’t see in fossils,” Hawks says. “It’s so much more than the bones that we have. It’s exciting for me because here’s this ancient group of people, and you’ve opened a new door into what their lives are like.” More than anything, he says, he just wants to get his hands on the data. “If you think about everything that’s been written on Neanderthals for the past 150 years … we have the potential to change everything.”

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Credit: Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology

Tagged: Biomedicine, DNA, genome, sequencing, genome analysis, Neanderthal

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