NEXT GENERATION SEQUENCING: P.292A new method for mtDNA deletion detection using massively parallel sequencing

Abstract

Human mitochondrial genome (mtDNA) is a circular DNA of 16 Mb in size. Mutations on mtDNA can cause several inherited diseases, many of which are neuromuscular. The mutational rate compared to nuclear DNA is very high. Clinically relevant mtDNA mutations are not only single nucleotide variants but also large deletions that can compass several thousand nucleotides. Because every cell contains several mitochondria, mtDNA mutations are usually heteroplasmic (1-99%). MtDNA mutations have been a methodological challenge because there has not been a method that could detect all mutation types. Especially estimating the heteroplasmic rate of mtDNA deletions has been challenging. We have analyzed 60 samples for mtDNA deletions. DNA was isolated from muscle. MtDNA was amplified using long range PCR followed by massively parallel sequencing (MPS). In addition, for five positive control samples mtDNA was enriched and the samples were sequenced without PCR amplification. The results were analyzed for all mutation types. The mtDNA deletions were detected using a new algorithm generated for the purpose. The results of mtDNA deletion analysis were confirmed by MLPA. Our method was able to detect all single deletions with great precision, with exact breakpoints of the deletions. Our new algorithm calculated the heteroplasmic rate correctly for smallest deletions but with very large deletions the MPS results were not fully concordant with MLPA. Our new method is an easy and comprehensive method for detecting all kinds of mtDNA mutations and calculating their heteroplasmic rate. The PCR amplification seems to create a bias that exaggerates the amount of large deletions. However, we are confident that this problem can be circumvented by replacing the PCR step with mtDNA enrichment.