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!
Finding conserved primer regions by aligning coding sequences has been increasingly common in comparative developmental studies over the past several years. Its not uncommon to find 40-100bp regions of 80-100% conservation at 5′ and 3′ ends of interesting genes making cloning from cDNA nearly trivial. But due to large gaps in the published chicken genome I often find it more useful to align sequences from chicken, zebra finch, turkey, green anole, mouse, and human as a first pass. All of these are available on Ensemble. In my experience, cloning ~30 genes from anoles, I find regions of high conservation between the mammalian and anole genomes that don’t even appear in the anole-chicken alignments. While I appreciate the additional difficulties of working with genomic DNA (finding primers that span introns for example) this may be another method of finding conserved primer regions that can work on additional species- in the off chance that the long list of primers supplied in the genome paper doesn’t provide systematists with enough work to keep themselves busy.
A draft assembly of the Burmese Python genome was also just publicly released a few weeks ago. The assembly is extremely fragmented, but is very high coverage (~17x). Primer design for squamates has become a snap!