Lemmon, along with his collaborator (and wife) Emily Moriarity Lemmon, an assistant professor in the Department of Biological Science at The Florida State University, decided six years ago to apply next generation sequencing technology to the study the phylogenetics of non-model organisms. But what they needed was a way to find common sequences in divergent genomes and isolate those for sequencing.
For their anchored enrichment approach, Lemmon used highly conserved anchor regions of vertebrate genomes as probes (2). Through a comparison of the genomes of five model vertebrates, these anchor regions were chosen for being highly conserved single-copy sequences flanked by less conserved regions that were well distributed throughout the genome. The level of conservation of these anchor regions, however, is lower than the UCEs used by Faircloth. This sequence variation is useful for studying shallower time scales.
Using their method, Lemmon's team was able to capture a substantial number of anchor sequences from each of five non-model organisms when its corresponding model organism in the same vertebrate clade was used to generate the bait probes. The divergence times between model and non-model organisms in each pair ranged from 94 to 254 million years.
Having established the utility of anchored hybrid enrichment, Lemmon now wants to share his lab's expertise and infrastructure. “We really wanted to make sure we built infrastructure and planned for the long term,” explains Lemmon. “We could have the sort of system set up so that anyone who wants to work on any species can either work with us or not, but we have really good tool kits for doing the probe identification and probe design, and then the downstream bioinformatics as well.” In the end, this approach is ideal for those without expertise in next-generation sequencing or who don't have the time and money to carry it out.
Based on word of mouth, Lemmon's group has undertaken about 12-16 collaborations in the past year. “The next year's going to be a big boom year for the anchored phylogenomics method. We have a lot of people that we're developing kits for which are going to turn into a lot of papers.”Scoring a Touchdown
While the UCE and anchored enrichment work well, each requires identifying and targeting highly conserved sequence elements in species whose genomes are otherwise highly divergent. This, however, is not ideal for targeting particular genes of interest that are highly divergent from the baits used for enrichment. It was this issue that prompted Gavin Naylor, a professor in the Department of Biology at the College of Charleston, to develop another approach to targeted enrichment sequencing for phylogenetic analysis (3), an approach that can isolate any large group of target genes in evolutionarily divergent organisms.
“We wanted to be able to choose parts of the genome that we thought were interesting and had good properties for phylogenetics. But we wanted to be able to choose our targets; we didn't want to be painted into a corner and only choose those which were ultraconserved,” says Naylor.
As it turns out, existing gene capture protocols are not suitable for targeting highly divergent gene sequences. “Gene capture is very specific and very stringent, and while you can pull it out of one species, you try to do it in another one, it's not going to work. So, that's where we try to use relaxing biochemistry protocols to pull them out.” Naylor's solution, as devised by Chenhong Li, his post-doc at the time, was to use a touchdown hybridization scheme involving lower stringency hybridization and washing steps for gene capture, thereby allowing the probes to retain more divergent target sequences.
To test their method, Naylor's group first selected target genes in several model vertebrates that were present as unique sequences in all of their genomes. After eliminating possible paralogs, these putatively orthologous sequences were used as baits in two rounds of gene capture with touchdown hybridization to isolate sequences from a distantly related species in the same vertebrate class for each of the model vertebrates. The divergence times for the species in each of these pairs varied from 100 to 300 million years, with similarities of target sequences ranging from 89% to 61%.
Compared to standard conditions, the two rounds of gene capture with the less stringent hybridization and wash conditions greatly increased the number of target sequences captured, and this improvement was especially dramatic when the target species were more highly divergent from the bait species.