The Establishment Of A High-throughout Sequencing Method For The Human Mitochondrial Genome

Mitochondria are cells' power plants present in almost every eukaryotic cell,which provide the capacity for aerobic respiration.They have the function of self-replicating,transcription and coding.In different cells,there are variable numbers of mitochondria from a few to several thousand,depending on the tissue type.The cells with higher metabolic activity will contain more mitochondria.The human mitochondrial genome is a double-stranded closed circular genome which is 16,569 bp in length.The human mtDNA consists of a non-coding mtDNA control region and a coding region encoding 13 proteins that critical to oxidative phosphorylation,as well as 22 tRNAs and 2 rRNAs.The non-coding region contains the origin of replication and transcription regulatory elements.The mtDNA is often utilized as a molecular marker in studies of population genetics,human evolution and forensic medicine due to the features of mtDNA including maternal inheritance,multiple copies,heteroplasmy,high evolutionary rate and so on.The mutation rate of mtDNA is higher.The repair system of mtDNA can't offset oxidative damage.A considerable amount of proofs supports influence of mtDNA mutation in some diseases such as Parkinsons disease,Alzheimers disease,diabetes,schizophrenia,cancer and so on.The recent years have seen many developments in sequencing.Next-generation sequencing(NGS)offers a high throughput solution for sequencing large amounts of mtDNA.Therefore the mitochondrial genome has been used quite widespread in broader areas.However,the process of library-building used commonly is expensive and complex.The mtDNA sequencing technique based on multiplex PCR is simpler and requires fewer samples,which can be applied to analyzing mtDNA mutation.Therefore,this master dissertation has intended to analyze sequencing technique based on the published studies,and build an easy,efficient,economical and stable human mtDNA high-throughput sequencing method.This study amplified the 96 Chines complete mitochondrial genomes using multiplex PCR.The whole mtDNA is covered by overlapping amplicons,which was sequenced on an Illumina HiSeq X Ten instrument.The results of this study showed that the mean depth of each base was more than 2,000×.100% mtDNA coverage was acquired across 96 samples with a mean depth of coverage threshold of 100×.This study established an optimal combination of primers and reaction system and built a human mtDNA massively parallel sequencing method based on multiplex PCR.The method we proposed is efficiency,simple and economical,is specific for Asian and can be applied to sequencing degraded DNA.The property of the method we built makes it the choice for studies of mtDNA,and contributes to delving into the complex genetic diseases,human evolution and forensic medicine.