Motivation: When working with non-model organisms, few if any species-specific markers are available for phylogenetic, phylogeographic and population studies. Therefore, researchers often try to adapt markers developed in distantly related taxa, resulting in poor amplification and ascertainment bias in their target taxa. Markers can be developed de novo and anonymous nuclear loci (ANL) are proving to be a boon for researchers seeking large numbers of fast-evolving, independent loci. However, the development of ANL can be laboratory intensive and expensive. A workflow is described to identify suitable low-copy anonymous loci from high-throughput shotgun sequences, dramatically reducing the cost and time required to develop these markers and produce robust multilocus datasets.
Results: By successively removing repetitive and evolutionary conserved sequences from low coverage shotgun libraries, we were able to isolate thousands of potential ANL. Empirical testing of loci developed from two reptile taxa confirmed that our methodology yields markers with comparable amplification rates and nucleotide diversities to ANLs developed using other methodologies. Our approach capitalizes on next-generation sequencing technologies to enable the development of phylogenetic, phylogeographic and population markers for taxa lacking suitable genomic resources.