New primers for sequencing nuclear loci from Anolis!
Availability of genomic loci for sequencing has long been a major stumbling block to evolutionary inference in non-model taxa. In anoles, for example, several decades of work relied almost exclusively on mitochondrial DNA. As part of the Anole genome sequencing initiative, my lab group collaborated with the Broad Institute to identify conserved primers that can be used to amplify nuclear loci from across Anolis. We ultimately tested 200+ primer pairs, most of which were identified by comparing the genome of Anolis carolinensis to genomic data from two related lizards (Anolis marmoratus and Polychrus marmoratus) and the chicken (others came from recent work in the Jackman lab). Through these comparisons, we identified conserved primers that could be used to amplify short genomic fragments (<1000 bp) sampled from across the genome. Using preliminary genome annotations, we specifically sought a genomically diverse sample including both exonic and non-exonic sequences. With the later addition of chromosomal data to the genome, we were able to show that the loci in our sample are also distributed across the genome, albeit primarily on the macrochromosomes (as one would expect based on their size relative to the microchromosomes).
After running PCR tests on anoles and other squamates, we attempted to obtain sequence data from across a two outgroups and Anolis for around 96 loci. This effort resulted in the 46 locus dataset included in the genome paper. Although this dataset has plenty of missing data, we found lots of primers that worked quite well across a broad spectrum of anole species. Some of the loci we sequenced are quite variable, and most are relatively easily aligned. All in all, we found a nice new sample of loci that are likely to be informative for both phylogenetic and population genetic studies. We’ll soon get more details out in a note we’re preparing for publication. In the mean time, our most successful primers are published in the on-line supplement to the genome paper. Those that we experimented with, but didn’t sequence extensively, can be acquired by dropping me a line – I’m more than happy to share.
Yes, next generation sequencing of non-model organisms is on the immediate horizon, but I think these new primers will prove useful for at least a few more years!