Objective To study the heteroplasmy of the whole mitochondrial genome genotyping result of hair shaft samples using HID Ion GeneStudio^TM S5 Sequencing System. Methods The buccal swabs and blood of 8 unrelated individuals, and hair shaft samples from different parts of the same individual were collected. Amplification of whole mitochondrial genome was performed using Precision ID mtDNA Whole Genome Panel. Analysis and detection of whole mitochondrial genome were carried out using the HID Ion GeneStudio^TM S5 Sequencing System. Results The mitochondrial DNA sequences in temporal hair shaft samples from 2 individuals showed heteroplasmy, while whole mitochondrial genome genotyping results of buccal swabs, blood, and hair samples from the other 6 unrelated individuals were consistent. A total of 119 base variations were observed from the 8 unrelated individuals. The numbers of variable sites of the individuals were 29, 40, 38, 35, 13, 36, 40 and 35, respectively. Conclusion Sequence polymorphism can be fully understood using HID Ion GeneStudio^TM S5 Sequencing system.
DNA, Mitochondrial/genetics* ; Genome, Mitochondrial/genetics* ; Heteroplasmy ; High-Throughput Nucleotide Sequencing ; Humans ; Sequence Analysis, DNA

Schizothorax argentatus that only distributes in the Ili River basin in Xinjiang is one of the rare and endangered species of schizothorax in China, thus has high scientific and economic values. In this study, the complete mitochondrial genome sequence of S. argenteus with a length of 16 580 bp was obtained by high-throughput sequencing. The gene compositions and arrangement were similar to those of typical vertebrates. It contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding region (D-loop). The nucleotide compositions were A (30.25%), G (17.28%), C (27.20%), and T (25.27%), respectively, showing obvious AT bias and anti-G bias. Among the tRNA genes, only tRNA-Ser^(GCU) could not form a typical cloverleaf structure due to the lack of dihydrouracil arm. The AT-skew and GC-skew values of the ND6 gene were fluctuating the most, suggesting that the gene may experience different selection and mutation pressures from other genes. The mitochondrial control region of S. argenteus contained three different domains, i.e., termination sequence region (ETAS), central conserved region (CSB-F, CSB-E, CSB-D, and CSB-B), and conserved sequence region (CSB1, CSB2, and CSB3). The conserved sequence fragment TT (AT) nGTG, which was ubiquitous in Cypriniformes, was identified at about 50 bp downstream CSB3. Phylogenetic relationships based on the complete mitochondrial genome sequence of 28 Schizothorax species showed that S. argenteus had differentiated earlier and had a distant relationship with other species, which may be closely related to the geographical location and the hydrological environment where it lives.
Animals ; Genome, Mitochondrial/genetics* ; Phylogeny ; Sequence Analysis, DNA ; Cyprinidae/genetics* ; RNA, Transfer/genetics* ; DNA, Mitochondrial/genetics* ; Genes, Mitochondrial

Sequence analysis of human mitochondrial DNA(mtDNA) is being used widely to characterize individual identification, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis. As sample decomposes, however, the possibility of mtDNA to be degraded becomes high and the possibility of spurious results becomes high. In this case mtDNA sequencing results must be carefully analyzed. We got unusual results while typing two human bone samples, which were not compatible with human mtDNA sequence. Bones were about 50 and 35 years old. We report the results with discussions about ancient DNA sequencing.
Adult ; DNA* ; DNA, Mitochondrial* ; Hair ; Humans ; Sequence Analysis ; Sequence Analysis, DNA ; Tooth

OBJECTIVETo investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM).

METHODSBlood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms.

RESULTSThe mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05).

CONCLUSIONThe mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.

Adult ; Complementarity Determining Regions ; genetics ; DNA Mutational Analysis ; DNA, Mitochondrial ; analysis ; Diabetes Mellitus, Type 2 ; genetics ; Female ; Genetic Predisposition to Disease ; Genome, Mitochondrial ; genetics ; Humans ; Male ; Sequence Analysis, DNA

OBJECTIVETo explore the application value of next generation sequencing (NGS) in the diagnosis of mitochondrial disorders.

METHODAccording to mitochondrial disease criteria, genomic DNA was extracted using standard procedure from peripheral venous blood of patients with suspected mitochondrial disease collected from neurological department of Beijing Children's Hospital Affiliated to Capital Medical University between October 2012 and February 2014. Targeted NGS to capture and sequence the entire mtDNA and exons of the 1 000 nuclear genes related to mitochondrial structure and function. Clinical data were collected from patients diagnosed at a molecular level, then clinical features and the relationship between genotype and phenotype were analyzed.

RESULTMutation was detected in 21 of 70 patients with suspected mitochondrial disease, in whom 10 harbored mtDNA mutation, while 11 nuclear DNA (nDNA) mutation. In 21 patients, 1 was diagnosed congenital myasthenic syndrome with episodic apnea due to CHAT gene p.I187T homozygous mutation, and 20 were diagnosed mitochondrial disease, in which 10 were Leigh syndrome, 4 were mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes syndrome, 3 were Leber hereditary optic neuropathy (LHON) and LHON plus, 2 were mitochondrial DNA depletion syndrome and 1 was unknown. All the mtDNA mutations were point mutations, which contained A3243G, G3460A, G11778A, T14484C, T14502C and T14487C. Ten mitochondrial disease patients harbored homozygous or compound heterozygous mutations in 5 genes previously shown to cause disease: SURF1, PDHA1, NDUFV1, SUCLA2 and SUCLG1, which had 14 mutations, and 7 of the 14 mutations have not been reported.

CONCLUSIONNGS has a certain application value in the diagnosis of mitochondrial diseases, especially in Leigh syndrome atypical mitochondrial syndrome and rare mitochondrial disorders.

Child ; DNA, Mitochondrial ; genetics ; High-Throughput Nucleotide Sequencing ; Homozygote ; Humans ; Leigh Disease ; Mitochondrial Diseases ; diagnosis ; Mitochondrial Encephalomyopathies ; Mutation ; Optic Atrophy, Hereditary, Leber ; Phenotype ; Point Mutation ; Sequence Analysis, DNA

The feasibility of species identification using sequence analysis of the cytochrome B (Cyt B) gene in mitochondrial DNA was investigated. DNA was extracted from nine different animals that could be easily met in our surroundings and Cyt B gene was amplified. Direct sequencing results for the amplified PCR products were compared with each other. Human was also included. Nucleotide sequence of the Cyt B gene for each animals was also compared with the previously known ones registered in nucleotide databases, Genebank. The inter-species sequence variation was high as the percent similarity of each sequences ranged 64.6-83.5%. Compared to this the percent similarity of sequences obtained here were high when compared to the sequences of the same species registered in the database showing relatively low intra-species variation. This data shows that the nucleotide sequences of Cyt B gene in a certain biological materials can be identified at species level. The applicability of this method to the forensic field is also demonstrated by performing a casework; determination of the origin for the placentae which were commercial available as "invogorant". Points about the use of Cyt B gene in forensic field was also reviewed.
Animals ; Base Sequence ; Cytochromes b* ; Cytochromes* ; DNA ; DNA, Mitochondrial ; Humans ; Placenta ; Polymerase Chain Reaction ; Sequence Analysis

The feasibility of species identification using sequence analysis of the cytochrome B (Cyt B) gene in mitochondrial DNA was investigated. DNA was extracted from nine different animals that could be easily met in our surroundings and Cyt B gene was amplified. Direct sequencing results for the amplified PCR products were compared with each other. Human was also included. Nucleotide sequence of the Cyt B gene for each animals was also compared with the previously known ones registered in nucleotide databases, Genebank. The inter-species sequence variation was high as the percent similarity of each sequences ranged 64.6-83.5%. Compared to this the percent similarity of sequences obtained here were high when compared to the sequences of the same species registered in the database showing relatively low intra-species variation. This data shows that the nucleotide sequences of Cyt B gene in a certain biological materials can be identified at species level. The applicability of this method to the forensic field is also demonstrated by performing a casework; determination of the origin for the placentae which were commercial available as "invogorant". Points about the use of Cyt B gene in forensic field was also reviewed.
Animals ; Base Sequence ; Cytochromes b* ; Cytochromes* ; DNA ; DNA, Mitochondrial ; Humans ; Placenta ; Polymerase Chain Reaction ; Sequence Analysis

For evaluation of the five coding region (CR) polymorphism in mitochondrial DNA (mtDNA); we had performed PCR and direct sequencing in 599 unrelated Korean who showed the identical DNA type in D-loop mitochondrial DNA analysis for total 2,810 bp fragment. Following the sequence analysis, all the sequences of five regions were compared respectively to Anderson standard sequence to investigate the nucleotide variations. The result showed, a total 4,565 nucleotide variations were observed at 190 positions in five CR as 3,931 (86.11%) substitutions, 32 (0.7%) insertions, and 602 (13.19%) deletions and the allele diversities (h) were higher than 0.9992 when adding each CR or combined CR to D-loop analysis in mtDNA. In conclusion, we could confirm the five CR are useful for forensic testing through the nucleotide variation and hapolotypes polymorphism.
Alleles ; Clinical Coding* ; DNA ; DNA, Mitochondrial* ; Polymerase Chain Reaction ; Sequence Analysis

BACKGROUND: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. METHODS: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. RESULTS: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. CONCLUSION: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.
Computational Biology ; DNA, Mitochondrial* ; Gene Frequency ; High-Throughput Nucleotide Sequencing* ; Methods ; Sequence Analysis, DNA

OBJECTIVETo establish a rapid, accurate, noninvasive and low cost method for screening MT3243A>G mutation in mitochondrial diabetes.

METHODSBlood, saliva, and urine sediment samples were collected from 6 patients with confirmed mitochondrial diabetes and 50 healthy controls from Shanghai Children's Hospital and Shanghai Sixth People's Hospital. The heterozygosity levels of MT3243A>G mutation in above samples were detected with pyrosequencing, and the data were compared. MT3243A>G mutations were rapidly screened with high resolution melting curve analysis (HRM) in the urine sediment samples of 1070 diabetic patients from 4 communities in Shanghai. Furthermore, pyrosequencing was used to validate the suspected positive samples, and the heterozygosity levels were also quantified.

RESULTSComparative experiments found that heterozygosity of MT3243A>G mutation was 2 to 7 times higher in urine sediment than in saliva and blood samples from the 6 patients with confirmed mitochondrial diabetes. However, the heterozygosity was slightly higher in saliva than blood samples. MT3243A>G mutation was not detected in the 50 healthy controls. Two samples with suspected MT3243A>G mutation were identified in the 1070 urine sediment samples of diabetes patients with HRM screening, which were validated by pyrosequencing. The heterozygosity of MT3243A>G mutation were 33.32% and 14.67% in the urine sediment samples, respectively.

CONCLUSIONUrine sediment samples can be used for rapid screening of MT3243A>G mutation for its ease to collect, noninvasiveness and higher level of heterozygosity. HRM is suitable for rapid screening for mitochondrian mutations for its low cost, while such mutations could be detected with sensitivity and accuracy by pyrosequencing.

DNA, Mitochondrial ; genetics ; Diabetes Mellitus ; genetics ; Heterozygote ; Humans ; Mutation ; Sequence Analysis, DNA ; methods ; Transition Temperature