Stepping in a Pile of…New Genomic Data

img_33031Genomic sequencing has made incredible strides in recent years, with both the cost and the time required to sequence an individual’s entire DNA sequence dropping meteorically. Yet one rate-limiting step for securing an organism’s genome remains: in order to sequence a species’ genetic information, you need a sample to start with. In humans or laboratory animals, a sample of blood or tissue is easily obtained. But what if a scientist wants to do a genomic study on an endangered species population, in the wild, without having to “trap 0r dart” a number of the animals to take blood samples?

George Perry, a genetics researcher at the University of Chicago, pondered this dilemma in planning his own research on endangered lemurs in Madagascar. In discussions with colleagues, he considered whether a “non-invasive” sampling technique might be possible for the collection of genomic data useful for conservationists and evolutionary biologists. The process led him to an unorthodox idea.

“We started thinking, ‘Is there a way to use fecal samples but to still do genomics work?’,” said Perry, a postdoctoral researcher in the laboratory of Yoav Gilad. “Then everyone would have the flexibility to collect population genomics data from any species at any time, as long as you can collect poop.”

Believe it or not, the collection of genetic data from feces has a long scientific history. Alongside the unwanted parts of an organism’s diet, solid waste contains a small number of cells stripped from the lining of the organism’s digestive system. Scientists have extracted small segments of DNA from those cells for study, mostly from the intracellular structures called mitochondria, which have their own genes. But more extensive genetic mapping of nuclear DNA from fecal samples has been thwarted by another of its ingredients: bacteria. The dominance of bacteria over host DNA inside the digestive system carries over to its product, where an organism produces less than 2% of the DNA deposited in its droppings.

To apply the awesome power of next-generation sequencing technology to a fecal sample, the DNA you want has to be separated from all that DNA you don’t want. Perry decided to modify an existing technique known as DNA capture (which has also been used to sequence Neanderthal DNA), to accomplish this task. With DNA capture, custom-made RNA sequences are used as bait to fish specific stretches of DNA out of a mixture; metallic beads are attached to the RNA sequences, and a magnet separates out the target DNA from the unwanted material. Perry boosted the specificity of this model, incorporating extra washes and two separate rounds of DNA capture, to turn his lower-quality fecal sample into starting material sufficient for sequencing. In part, that means starting with a lot more DNA that typically used for DNA capture, which means starting with roughly 2 grams of poop from each animal. Fortunately, it’s an abundant resource.

“It’s not that you can only study rhinoceros because they have huge poop,” Perry said.

Once the purification was worked out, Perry and his colleagues John Marioni, Pall Melsted, and Yoav Gilad were able to pit fecal samples against blood samples (both taken from captive West African chimpanzees) to see if the quality of his new source measured up to the gold standard. The results, published earlier this month in Molecular Ecology, were hardly crap: in terms of sequence accuracy, the two samples were almost identical. That gives Perry the confidence to use the technique out in the field for studying variation in animal populations.

“In humans, we have a number of examples where we have learned about the evolutionary history of our species from population genetic analyses,” Perry said. “With this kind of genomic scale sequencing approach, we will be able to do similar analyses in endangered species, to understand their evolutionary history and ecology, how they’re adapted to interact with the environment. If we understand that, then we can make better decisions on conservation and make sure certain things in their environment that are critical for their survival don’t go away.”

In an ideal protocol, a researcher will only have to trap or dart one or two animals from a species, or obtain a blood sample from a member of the species in a zoo. Those blood samples would be used to create a high-quality genome reference that can be broken down into new “baits” for the DNA capture technology. Then researchers could collect the droppings of hundreds of individuals, and each could be sequenced to measure diversity within a population or between populations.

“It requires one high-quality DNA sample, and I think in many cases it’s justifiable to trap one animal from a species,” Perry said. “But if there’s only 3,000 animals left of a species, I don’t want to dart 100 of them.”

Perry will put his new method to work in Madagascar, where the arrival of humans 2,500 years ago produced massive changes in the population of native lemurs. Perhaps due largely to hunting by humans and habitat disruption, large lemur species went extinct, and the smaller species that still exist today may have been forced to adapt because of changes to their environment. By studying the genetic variability in today’s Madagascar lemurs, Perry can draw conclusions about how they managed to survive the human invasion and how their population sizes have changed over time, and use that information to help their survival against ongoing challenges from our continued presence in their environment.

It’s a noble goal, made possible by realizing the genomic potential of an ignoble substance. Perry himself doesn’t mind the messy nature of his new research material, even coming up with the hook for a rap about his new method to capture valuable DNA from a fecal sample: “I can bind what comes out of your behind.”

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Perry GH, Marioni JC, Melsted P, & Gilad Y (2010). Genomic-scale capture and sequencing of endogenous DNA from feces. Molecular ecology PMID: 21054605

About Rob Mitchum (526 Articles)

Rob Mitchum is communications manager at the Computation Institute, a joint initiative between The University of Chicago and Argonne National Laboratory.

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