Primer designing strategy for amplification and sequencing of the complete mitochondrial genome of Semnopithecus hypoleucos

Abstract

The mitochondrial genome is highly informative for evolutionary analysis of organism lineages and phylogenetic studies. The availability of robust primers for amplifying complete mitochondrial genomes is a crucial step in current mitogenome studies. However, organism specific characteristics such as variable transition to transversion substitution ratios seen in some groups pose challenges for the development of universal, or at least broadly applicable, primer pairs for this purpose. This study reports on a strategy of primer design and optimization (PDO) where regions of known mtDNA genescan be used for choosing primers for amplification, sequencing and assembly of entire mitochondrial genomes of several closely-related species. In brief, taking advantage of the circular organization of mtDNA, primers are first designed for amplification of “long” products using the 5’ region of one conserved gene and a 3’region from another conserved gene. Additional primers are then used to amplify “short” regions to fill in gaps to allow for complete assembly of the genome. We show how we were able to use this approach to successfully amplify entire mitochondrial genomes from a non-human primate species (Semnopithecus hypoleucos), and also how this provided data useful for annotation of the assembled genome data.