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Special News Feature - DNA Sequencing
 
Patrick C. H. Lo, Ph.D.
BioTechniques, Vol. 55, No. 4, October 2013, pp. 175–177
Full Text (PDF)



For decades, systematic biologists have been defining evolutionary relationships amongst all organisms and creating phylogenetic “trees” to illustrate their findings. But delineating the exact shape and branching patterns along the so-called “Tree of Life”—the phylogenetic tree describing the evolutionary relationships of all organisms—remains a challenge, even in this era of molecular biology where deep sequencing and genomic analysis are commonplace.

Until recently, classifying an organism along a phylogenetic tree required PCR amplification with degenerate primers followed by amplicon sequencing to study genomic loci in distantly related taxa. This approach, however, limits the number of loci that can be simultaneously examined to significantly less than the hundreds or thousands needed to truly classify very distant taxa.

In order to overcome these limitations and enhance the resolution and size of the Tree of Life, several groups are advancing new methods, combining the power and throughput of next-generation sequencing with novel targeted enrichment strategies to easily capture and sequence thousands of loci in a single experiment.

Ultraconservative

Targeted enrichment uses specially designed oligonucleotide probes to isolate specific sequences from a mixture of DNA molecules through hybridization either on a solid surface or in solution. Isolated sequences can then be used for downstream analyses, including next-generation sequencing. Although developed initially to pull out coding sequences from closely related individuals, evolutionary biologists quickly saw huge potential for targeted enrichment in studies of distantly related species.

“Targeted enrichment techniques allow you to target many hundreds of thousands of loci very easily. What we then needed was a target to go after, and conserved sequences distributed throughout the genome are a good target,” notes Brant Faircloth, an assistant researcher in the Department of Ecology and Evolutionary Biology at UCLA. Faircloth and his colleagues decided to extend targeted enrichment techniques by using ultraconserved elements (UCEs) as probes. Originally discovered in humans, UCEs are somewhat mysterious sequences with unclear functions whose high sequence conservation and other features make them well suited as molecular markers.

Using around 2400 UCE-anchored loci from 9 non-model avian species, Faircloth et al. were able to obtain alignments of nearly 850 loci, recovering the established phylogeny among and within 3 bird lineages that had diverged by 65 million years, providing one of the first demonstrations that next-generation sequencing of UCE-enriched DNA could be applied across the Tree of Life on a fairly deep time-scale.

While his methodology can work across deep time scales, Faircloth actually intends to reverse gears and apply the technique to shallower time-scale questions.

“Phylogeographic studies are of interest to us. We want to start looking at populations of individuals and how those populations may have diverged over shorter timespans. As opposed to 200-400 million years ago, now we're talking about 10-30 million years ago and less,” explains Faircloth. In addition, the method will be used for population studies. “Among individuals, within populations, we know the same suite of loci will enrich targets that are informative at the individual level. We can actually look at parents and offspring within a population and infer who the parents of offspring are, using these same exact ultraconserved loci that we've targeted for phylogenetic questions.”



Not Quite So Ultra

Annoyance, in part, fueled Alan Lemmon's development of a targeted enrichment sequencing method. “[We] were fed up with having to develop primer pairs for each new non-model species we were working on, or at least getting existing primer pairs working,” recalls Lemmon, an assistant professor in the Department of Scientific Computing at The Florida State University.

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