Laser Capture Microdissection and Whole Chromosome Amplification for Sequencing Large Genomes

The Mexican axolotl (Ambystoma mexicanum) diverged along with the amphibian lineage from other tetrapods approximately 300 million years ago. Even with a massive genome ~30Gb, the axolotl only has 14 haploid chromosomes, and its gene orders are highly conserved with reptilian and mammalian genomes. As such, the Ambystoma is an important model organism for studying evolution of vertebrate genome changes and could provide information pertaining to the changes in gene order, chromosome structure and genome size in vertebrates. We are currently attempting to construct a complete genome assembly for the salamander, building on our existing linkage map.

Due to current limitations on assembling large genomes with many repeats and lengthy introns, an alternative approach was developed; sequencing one chromosome at a time. Single dyads were excised from Ambystoma chromosome spreads via laser capture microdissection (LCM), amplified, and then sequenced (Illumina HiSeq). The resulting reads from chromosome-specific libraries were aligned to mapped Ambystoma sequences and covered more than 80% (75/92) of known genes on the targeted chromosome, with high fidelity to linkage group 3.

These findings indicate that our LCM based approach yields robustly targeted sequencing, which will, in the short term, lead to modification of the current linkage map, and ultimately provide data for a future assembly. These preliminary results prove our method for obtaining whole genome sequence for the salamander and production of a whole genome assembly for this valuable model organism, providing a wealth of insights into vertebrate genome evolution, sex chromosome evolution and tissue/organ regeneration.