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Array-Based Capture Method Helps Target and Sequence Highly Contaminated Neandertal DNA

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By Monica Heger

This story was originally published May 6

Researchers have used a targeted method to capture, amplify, and sequence highly contaminated Neandertal DNA on the Illumina Genome Analyzer. The method, published last week in Science, allowed them to identify changes to the human genome that have occurred since modern humans diverged from the Neandertal.

The team designed an oligonucleotide array using Agilent reagents to capture and sequence nearly 14,000 non-synonymous substitutions identified by whole-genome sequencing of the Neandertal to have changed on the human lineage since the last common ancestor shared with chimpanzees.

The resequencing project, performed by researchers at Cold Spring Harbor Laboratory, was done in collaboration with an international team led by Svante Pääbo's laboratory at the Max Planck Institute, which published the draft sequence of the Neandertal in last week's Science (see related story, same issue).

The team at Cold Spring Harbor, led by Greg Hannon, developed the capture method, and Pääbo's lab in Germany provided the ancient DNA samples.

Hannon had previously demonstrated that the capture method works especially well on highly contaminated DNA, but the Neandertal samples were thought to be unusable because they were 99.8 percent contaminated, mainly with microbial DNA.

The researchers used hybridization capture on glass slide microarrays with Agilent reagents. They were able to recover over a megabase of the targeted regions, which included around 14,000 protein-coding regions where the human genome is known to differ from the chimpanzee genome. They designed 60-base probes to target the regions of interest and 50 bases on either side. Additionally, they included probes to estimate the level of contamination by modern human DNA.

They began with 454 libraries created by Pääbo's team, but before sequencing, converted them to be compatible with Illumina. They used a paired-end sequencing strategy with read lengths mostly around 50 base pairs. They were able to target 13,250 of the regions with an average 4.8-fold coverage.

By comparing the regions to the human reference genome, the chimpanzee genome, and human genomes from the Human Diversity Panel, they found that of the 14,000 non-synonymous substitutions where humans and chimps are different, only 88 are different between modern humans and Neandertals.

"I was pretty shocked that we were that similar," said Hannon. "And we've still only looked at one Neandertal. I think that number might get chipped away at even more" if the team examines more Neandertals.

The next step is to figure out the functional significance of those 88 changes. Hannon said that there are some clues that they could be major differences. For instance, in many cases they encode for "more radical amino acid changes with respect to the chemical properties of the amino acids" than in the other non-synonymous substitutions that were sequenced, according to the paper. Also, the 88 substitutions, which occur in 83 genes, "tend to affect amino acid positions that are more conserved than the older substitutions," the authors wrote.

Hannon said the method will be useful for studying any contaminated DNA, including ancient DNA and possibly some forensic samples. Additionally, it could be used to study evolution and to characterize variation even among highly divergent related genomes. As an example, he said that researchers could use an organism like the wallaby to study marsupials.

Hannon said he plans to continue to use the method to look at regions of variation and obtain deeper information about cancer genomes and DNA methylation states.

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