Optimization of whole mitochondrial genome sequencing with Ion Personal Genome Machine (PGM) System.

The human mitochondrial DNA (mtDNA) phylogeny is an almost perfect molecular prototype for a non-recombining locus and knowledge on its variation has been extensively used in molecular anthropology and population genetics studies. Furthermore, its mode of inheritance, high mutation rate and high cellular copy number make this locus a primary choice also in the field of ancient DNA analysis, as well as in different contest such as biomedical research and diagnostic of mitochondrial disorders or human identification for forensic purposes. In the very last years, the study of mtDNA control region sequence and coding region single nucleotide polymorphisms have been gradually substituted by the investigation of variation at the whole mitochondrial genome, allowing analyses at a much higher resolution level and a deeper discrimination between haplogroups. In fact, high-throughput sequencing of whole mitochondrial genomes is now possible with lower costs with respect to those related to the traditional Sanger method thanks to Next Generation Sequencing (NGS) technologies. Among them, the Ion Personal Genome Machine (PGM, Life Technologies) is a new platform that uses an innovative method of sequencing. In fact, it does not use fluorescence or chemiluminescence, but measures the proton (H+) ions released during base incorporation. This new sequencing method allows to reduce times and costs of sequencing. The workflow, like other NGS, consists of four major steps: library construction, template preparation, sequencing and analysis. We develop a workflow for processing multiple samples in parallel through the preparation of barcoded libraries obtained by physical fragmentation of two long range PCR products with Bioruptor sonication system (Diagenode) at 200 base pairs reads. After equalization of single barcoded libraries in a pool the samples are subjected to an emulsion PCR reaction step. In this step, the library are attached to beads and amplified. During a step of enrichment only the beads that bind amplified template fragments are retrieved and these will be the template for the sequencing reaction. The sequencing technology is what really differentiates Ion Torrent platform; since optics are not required, the sequencing reaction is relatively fast and inexpensive. The obtained sequences are processed and aligned to the reference genome by means of the Ion Torrent suite and a dedicated bioinformatics tool was used to call single nucleotide and small insertion/deletion variants. We compared our results with those obtained by processing raw data via an ad hoc developed pipeline, as well as with those resulting from Sanger sequencing of the same samples to assess the reliability of the adopted protocol and sequencing technology.