Mito-SiPE: A sequence-independent, PCR-free mitochondrial DNA enrichment method for ultra-deep sequencing that minimises amplification and alignment artefacts for the analysis of mitochondrial heteroplasmy/variation

Abstract

Background: The analysis of somatic variation in the mitochondrial genome requires deep sequencing of the mitochondrial genome. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. Results: Here, we demonstrate a method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome by selectively purifying the organelle with differential centrifugation and alkaline lysis of seven different mouse tissues. This method yields a preparation of highly enriched mtDNA and avoids the pitfalls inherent in the widely employed sequence dependent methods. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mtDNA enrichment. Discussion: Mitochondrial DNA from the un-adapted version of this method did not undergo quantification or short-read sequencing when it was initially reported. Here, we have described a modified version of the protocol and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are falsely identified as heteroplasmy when long-range PCR is used.