Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system.
Mitochondria are the “powerhouses” of human cells and disturbances in mitochondrial functions have been implicated in a wide range of human diseases, including cancer, heart disease, diabetes, Alzheimer's disease, and Parkinson's disease. The human mitochondrial genome, which is a circular DNA molecule that consists of 16,569 bp, encodes 13 polypeptides that are components of the electron transport chain (ETC), as well as 22 tRNAs and two rRNAs that contribute to mitochondrial protein synthesis. A variety of human diseases are directly associated with mitochondrial DNA (mtDNA) mutations and hundreds of putative pathogenic mtDNA variants have been identified.Mitochondrial DNA is present in hundreds to thousands of copies per cell and also has a very high mutation rate. New mtDNA mutations arise in cells, coexist with wild-type mtDNAs (heteroplasmy), and segregate randomly during cell division. The vast majority of deleterious mtDNA point mutations are heteroplasmic and their mutant load can vary significantly among different tissues, even in the same subject. Moreover, different percentages of mutant mtDNA can be associated with completely distinct clinical manifestations. Currently, it is challenging to identify all of mutations in the mitochondrial genome and simultaneously quantify the mtDNA heteroplasmy levels. In addition to the molecular diagnosis of mitochondrial diseases, there is a rapidly growing need for methods to analyze mtDNA variants for other applications, including evolutionary and forensic studies. Therefore, it is critical that mitochondrial genome sequences can be acquired and detected in a reliable, high-throughput, and cost-effective manner, especially in samples with clinically relevant levels of mtDNA heteroplasmy.
Currently, the two most popular complete mitochondrial genome sequencing methods are direct sequencing and the MitoChip. However, these two methods are neither sensitive nor specific enough to detect mtDNA heteroplasmy. Methods used for mitochondrial genome–wide heteroplasmic position screening include denaturing HPLC , Surveyor Nuclease digestion, and high-resolution melt (HRM) profiling . Although these methods can be used to detect mtDNA heteroplasmy, they cannot localize or quantify the heteroplasmic position(s). Several other techniques have been developed for the specific quantification of mtDNA heteroplasmy levels. These methods include PCR-RFLP analysis, allele-specific oligonucleotide dot-blot analysis, real-time amplification refractory mutation system quantitative PCR , and pyrosequencing. However, these methods are labor-intensive and can only be used to analyze a known mutation.
Recently developed parallel sequencing methods have the capacity for massive sequencing and offer a highly robust and less labor-intensive approach to genome-wide sequencing. Currently, there are four next-generation sequencing platforms: the Illumina Genome Analyzer (GA; San Diego, CA, USA), the Roche 454 Genome Sequencer FLX system (Indianapolis, IN, USA), the Applied Biosystems SOLiD system (Foster City, CA, USA), and the Helicos True Single Molecule Sequencing system (Cambridge, MA, USA). The small size of the human mitochondrial genome and the resulting high coverage for each nucleotide position generated by parallel sequencing should enable the detection of low levels of mtDNA heteroplasmy. Previously, 454 sequencing was used to generate 34.9-fold coverage of the mtDNA from ~0.3-g bone of a 38,000-year-old Neanderthal individual. The Illumina GA, coupled with target microarray-based capture, was successfully employed to re-sequence the entire mitochondrial genome (coverage >2,900) and the exons of 362 nuclear genes encoding mitochondrial proteins. However, neither of these studies investigated the capability of the technologies for heteroplasmy identification and quantification. In the current study, They utilized the Illumina GA system to sequence the entire human mitochondrial genome and determined the sensitivity and specificity of this platform for the analysis of heteroplasmic mtDNA samples.
Mitochondria are the “powerhouses” of human cells and disturbances in mitochondrial functions have been implicated in a wide range of human diseases, including cancer, heart disease, diabetes, Alzheimer's disease, and Parkinson's disease. The human mitochondrial genome, which is a circular DNA molecule that consists of 16,569 bp, encodes 13 polypeptides that are components of the electron transport chain (ETC), as well as 22 tRNAs and two rRNAs that contribute to mitochondrial protein synthesis. A variety of human diseases are directly associated with mitochondrial DNA (mtDNA) mutations and hundreds of putative pathogenic mtDNA variants have been identified.Mitochondrial DNA is present in hundreds to thousands of copies per cell and also has a very high mutation rate. New mtDNA mutations arise in cells, coexist with wild-type mtDNAs (heteroplasmy), and segregate randomly during cell division. The vast majority of deleterious mtDNA point mutations are heteroplasmic and their mutant load can vary significantly among different tissues, even in the same subject. Moreover, different percentages of mutant mtDNA can be associated with completely distinct clinical manifestations. Currently, it is challenging to identify all of mutations in the mitochondrial genome and simultaneously quantify the mtDNA heteroplasmy levels. In addition to the molecular diagnosis of mitochondrial diseases, there is a rapidly growing need for methods to analyze mtDNA variants for other applications, including evolutionary and forensic studies. Therefore, it is critical that mitochondrial genome sequences can be acquired and detected in a reliable, high-throughput, and cost-effective manner, especially in samples with clinically relevant levels of mtDNA heteroplasmy.
Currently, the two most popular complete mitochondrial genome sequencing methods are direct sequencing and the MitoChip. However, these two methods are neither sensitive nor specific enough to detect mtDNA heteroplasmy. Methods used for mitochondrial genome–wide heteroplasmic position screening include denaturing HPLC , Surveyor Nuclease digestion, and high-resolution melt (HRM) profiling . Although these methods can be used to detect mtDNA heteroplasmy, they cannot localize or quantify the heteroplasmic position(s). Several other techniques have been developed for the specific quantification of mtDNA heteroplasmy levels. These methods include PCR-RFLP analysis, allele-specific oligonucleotide dot-blot analysis, real-time amplification refractory mutation system quantitative PCR , and pyrosequencing. However, these methods are labor-intensive and can only be used to analyze a known mutation.
Recently developed parallel sequencing methods have the capacity for massive sequencing and offer a highly robust and less labor-intensive approach to genome-wide sequencing. Currently, there are four next-generation sequencing platforms: the Illumina Genome Analyzer (GA; San Diego, CA, USA), the Roche 454 Genome Sequencer FLX system (Indianapolis, IN, USA), the Applied Biosystems SOLiD system (Foster City, CA, USA), and the Helicos True Single Molecule Sequencing system (Cambridge, MA, USA). The small size of the human mitochondrial genome and the resulting high coverage for each nucleotide position generated by parallel sequencing should enable the detection of low levels of mtDNA heteroplasmy. Previously, 454 sequencing was used to generate 34.9-fold coverage of the mtDNA from ~0.3-g bone of a 38,000-year-old Neanderthal individual. The Illumina GA, coupled with target microarray-based capture, was successfully employed to re-sequence the entire mitochondrial genome (coverage >2,900) and the exons of 362 nuclear genes encoding mitochondrial proteins. However, neither of these studies investigated the capability of the technologies for heteroplasmy identification and quantification. In the current study, They utilized the Illumina GA system to sequence the entire human mitochondrial genome and determined the sensitivity and specificity of this platform for the analysis of heteroplasmic mtDNA samples.
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