Massively parallel sequencing (MPS) assays for sequencing mitochondrial genomes: the phylogenomic implications for Acropora staghorn corals (Scleractinia; Acroporidae)

Based on only a handful of mitochondrial and nuclear loci, the phylogenetic relationships within the genus Acropora have been unclear. However, with the new sequencing technology of massively parallel sequencing (MPS), the inter-specific relationships within Acropora may be resolved. We performed multiplex sequencing of the mitochondrial genome of eleven Acropora species representing different groups using the Illumina Solexa platform. Mitochondrial genomes were sequenced from long PCR-amplified templates ligated with different index sequences (similar to 9 kbp) and analyzed using the mitochondrial genome of Acropora tenuis as a reference. A total of 75 million read outputs in one Illumina lane were obtained, with mapping results having coverage up to 44,000-fold. Assembly results of multiplex samples confirmed with Sanger sequencing produced < 0.03 % error. Aligning the eleven mitochondrial genomes with the reference sequence revealed only 110 phylogenetically informative sites over the mitochondrial genome. The largest pairwise genetic distance observed was in the putative control region (0.022). A comparison of two phylogenetic trees based on the whole mitochondrial genome and control region showed that the former tree produces a higher resolution of phylogenetic relationships. In this study, we demonstrated the first case of sequencing cnidarian mitochondrial genomes by using multiplex MPS and applying it in a phylogenomic analyses.