Characterization of a Large Vertebrate Genome Using Shotgun and Laser Capture Chromosome Sequencing - Salamander

The Mexican axolotl (Ambystoma mexicanum) is an important amphibian model for several fields of biology, including biomedicine, regeneration and genome evolution. The axolotl genome has gene orders that are highly conserved with those of reptilian and mammalian genomes and provides valuable information for reconstructing several features of ancestral tetrapod genomes. However, the genomes of all other salamanders (including axolotl) are greatly expanded in size relative to other extant tetrapods. As such, the axolotl genome provides both a unique perspective on the evolution of genome structure/biology and presents a major challenge toward the development of genomic resources.

As part of a multipronged approach to sequence and assemble this large genome, we developed approaches to capture, amplify and sequence single chromosomes (dyads). Initial sequence analyses revealed that these libraries provide high sensitivity and specificity for individual chromosomes. These analyses both validate and extend our earlier linkage mapping studies. Results from ongoing efforts to assemble sequences from these laser capture libraries indicate that informative assemblies can be generated from a relatively small number of libraries (1-4). Altogether these data support the use of laser capture sequencing as part of a multi-faceted strategy for development of a high quality genome assembly for A. mexicanum and other large genomes.

 Our pilot studies suggest that these laser-capture sequencing methods will be generally applicable to a broad range of biological questions, including chromosomal scaffolding of genomes for organisms that are not amenable to laboratory culture and genomic characterization of microscopically identifiable cells (e.g. cancer or germ cells).