A team of researchers, led by College of Charleston Biology Professor Gavin Naylor, has developed a new targeted gene capture method to isolate and compare protein-coding genes across highly divergent vertebrate species in a single next-generation sequencing experiment. This new approach should allow biologists to more rapidly understand how organisms acquire new traits, such as disease predisposition or environmental adaptation, during evolution.
In an article appearing in the June 2013 issue of BioTechniques, the group reports that by using a combination of relaxed gene capture conditions and a second round of targeted capture, eight times as many genes could be isolated from different species when compared with existing protocols.
“We knew this was possible because of Southern blot technology. More than 35 years ago Ed Southern developed a way to bind and identify DNA sequences that are not perfectly matched, by adjusting stringency conditions,” says Gavin Naylor, professor of biology at the College of Charleston and senior author of the new paper.
Most targeted gene capture protocols in use today are designed to precisely target and accurately bind genomic regions that share greater than 95% identity to the sequence of a DNA bait. While this stringency is useful for capturing pre-specified sets of genes from a single species, it hampers attempts to study the same set of genes when they have diverged across multiple species.
Previous efforts to study cross-species genetic variation have focused on finding ultra conserved genetic elements that have not changed much during evolution and then sequencing neighboring genes for comparison.
“These are powerful tools, but what if the gene that you’re interested in doesn’t happen to be next to a conserved element? Then you’re out of luck,” explains Naylor.
With their new approach, Naylor and colleagues were able to target highly divergent genes for capture, regardless of their location in the genome. “We’re basically relaxing stuff, making it more sloppy, dialing down the precision,” says Naylor.
For more than a year, first author Chenhong Li, a former postdoctoral scholar in Naylor’s lab who is currently a professor at Shanghai Ocean University in China, tested a wide range of different gene capture conditions. By lowering the temperature for the final washing steps and adding a second round of gene capture using the genes captured during the first round as target samples, Li was able to dramatically improve the results. The modified protocol enabled capture of genes that differ by as much as 40% in DNA sequence—for some genes, this equates to divergence times of up to several hundred million years.
“Of course, there are some elements that we will not be able to get with this method, but that is both a strength and a weakness. If the technique captures everything that it randomly picks up from the genome, then you don’t know what you have,” says Naylor.
The robustness of the method allows researchers to study genes that are evolving slowly across distantly related groups of species, as well as genes that are evolving quickly across closely related species. For example, Naylor’s lab is particularly interested studying the evolution of chondrichthyan fishes that have transitioned from saltwater to freshwater environments. Some species of stingrays, sharks, and sawfishes have made this evolutionary leap, which is likely accompanied by changes in the genes associated with osmoregulation. By making capture baits for those genes, his team hopes to see if the different lineages have adapted to their freshwater environments in the same way.
“Hopefully, researchers will use this technology to quickly screen hundreds of different species for a particular genetic architecture of interest in a single next-generation sequencing run,” says Naylor.