1.Full Sibling Testing Based on NGS-SNP Genotyping Method and IBS Strategy.
Zhi Yuan WANG ; Di Jia WANG ; Ran LI ; Hai Xiao LI ; Na Na WANG ; Hong Yu SUN
Journal of Forensic Medicine 2019;35(2):205-209
Objective To evaluate the effectiveness of single nucleotide polymorphism (SNP) genoty-ping in combination with identity by state (IBS) strategy in full sibling testing. Methods Thirty-five blood samples were collected from a four-generation family. Ninety autosomal SNPs were genotyped using Precision ID Identity Panel. The distribution of IBS scores for full siblings and other relationships were calculated and compared. The relationships were determined using Fisher discriminant function and threshold method, respectively. Results Based on family members and previous research, 44, 30, 111, 71 and 1 000 pairs of full siblings (FS), grandparent-grandchild (GG), uncle/aunt-nephew/niece (UN), first cousins (FC) and unrelated individuals (UI) were obtained, respectively. The average IBS scores were 148, 130, 132, 124 and 120, respectively. Except for the GG and UN pairs, the distribution differences among the other relationships had statistical significance (P<0.05). The false rates of Fisher discriminant function to determine relationships were 1.3%, 22.3%, 17.0% and 38.7% for FS, GG, UN and FC, respectively. Based on the simulation data, the thresholds t1=128 and t2=141 were recommended to determine full sibling relationships (the false rate ≤0.05%). Conclusion The 90 SNP genetic markers included in the Precision ID Identity Panel meet the testing requirements for full sibling relationships. The threshold method based on IBS has a relatively lower false rate and is more flexible.
Genotype
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Genotyping Techniques/methods*
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Humans
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Polymorphism, Single Nucleotide/genetics*
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Siblings
2.Application of SNPstream for medical genetics research.
Li-juan FEI ; Lin-dan JI ; Li-na ZHANG ; Shuai HUA ; Jie-qiong JIN ; Jin XU
Chinese Journal of Medical Genetics 2012;29(1):9-12
OBJECTIVETo introduce the principle, procedure, efficacy and application of SNPstream genotyping technology.
METHODSGenotyping results of 152 SNPs were used to analyze the feasibility, call rate and accuracy of SNPstream technology.
RESULTSFor the 152 selected SNPs, 122 SNPs can be genotyped with SNPstream, for which 116 SNPs were successfully genotyped. Replication study showed that the repeatability of genotyping is 99%. When the allele cluster was clear, the accuracy can reach 100%. But when the allele cluster was obscure, the accuracy was only 93.8%.
CONCLUSIONSNPstream technology has the advantages of high accuracy, flexible throughput, and high cost performance, and may have a wide application for medical genetics research.
Alleles ; Genetics, Medical ; methods ; Genotyping Techniques ; methods ; Humans ; Polymorphism, Single Nucleotide ; genetics ; Reproducibility of Results
3.Genotype analysis and personalized medicine.
Chinese Journal of Pathology 2011;40(10):651-654
4.High resolution melting and its application in identity study of traditional Chinese medicine.
Kang CHEN ; Chao JIANG ; Yuan YUAN ; Lu-qi HUANG
Acta Pharmaceutica Sinica 2015;50(12):1581-1588
High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.
DNA Mutational Analysis
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methods
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Drugs, Chinese Herbal
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classification
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Genotyping Techniques
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methods
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Medicine, Chinese Traditional
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Nucleic Acid Denaturation
5.Identification and genotyping of difficult blood groups in the patients with phymatosis.
Chao WANG ; Su-Ping LI ; Min LI ; Xue-Zhong WU ; Xin XING ; Rong LYU
Journal of Experimental Hematology 2013;21(3):780-784
In part of the patients with blood disease or malignant tumors, especially those with leukemia and multiple myeloma, the disease state and unsuitable treatment often resulted in the inconsistence between positive and negative ABO blood group, displaying attenuation of the antigen or antibody of ABO blood group. This study was purposed to analyze the course of inconsistence between positive and negative ABO blood group and to perform the correct typing of erythrocytes and genes. The serology, absorption and elution test were used to examine the 12 tumor patient of the inconsistence between positive and negative typing. The 6th, 7th exon and 5-7th introns were amplified by PCR for questionable samples, and the gene sequencing of exon was performed. The results showed that 9 specimens were determined as 6 of A group, 2 of O group, 1 of B group, 3 cases were identified as O46, B108, and A102 group, respectively, by the serology, absorption and elution typing. The genotype of 2 cases among them was not identified because of the erroneous PCR amplified result or the contradicted sequencing results, failing to determine the ABO genotype. It is concluded that the serological method for blood grouping, genotyping, absorption and elution method can be used for the blood samples unable to typing because of the inconsistence between positive and negative typing of ABO group, therefore, guaranteeing the safety and effectiveness.
ABO Blood-Group System
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genetics
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immunology
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Blood Grouping and Crossmatching
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methods
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Genotype
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Genotyping Techniques
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methods
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Humans
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Neoplasms
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genetics
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immunology
6.Construction and application of a multiplex PCR system for genotyping of human red blood cell antigens.
Huan-huan GAO ; Yun-lei HE ; Lu-yi YE ; Pan WANG ; Zhong-hui GUO ; Zi-yan ZHU ; Yong-ming ZHU
Chinese Journal of Medical Genetics 2012;29(5):547-552
OBJECTIVEA reliable method for genotyping blood group antigens Dib, k, Jsb1910 and Jsb2019 was developed. Through screening for rare blood types, the National Rare Blood Bank of China may be enriched.
METHODSThe controls for allele detection of blood groups Dib, k, Jsb1910 and Jsb2019 were prepared via polymerase chain reaction (PCR)-mediated gene site-directed mutagenesis (SDM) technique. Sequence-specific primers were designed according to known single nucleotide polymorphism (SNP) sites of alleles of blood groups antigens Dib, k, Jsb1910 and Jsb2019, a multiplex PCR system was developed by optimizing PCR reaction system. And 4190 random healthy donors samples were screened for the blood group antigens.
RESULTSUsing SDM technique, controls for alleles in blood group Dib, k, Jsb1910 and Jsb2019 were successfully generated. And a multiplex PCR system for genotyping above blood groups was developed. After verification, the system has performed with good stability and reproducibility. Two Di (b-) samples have been discovered from 4190 samples, no k- and Js(b-) sample was found.
CONCLUSIONMultiplex PCR features rapid detection, high throughput and low cost, and can be used for screening for donors of rare blood types. Information of donors may be registered in a database, which in turn can help those with rare blood types or require long-term blood transfusion to obtain matched blood, thereby reduce the adverse reactions of blood transfusion.
Blood Group Antigens ; genetics ; Erythrocytes ; immunology ; Genotyping Techniques ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Mutagenesis, Site-Directed ; Polymorphism, Single Nucleotide
7.Establishment of the multiplex genotyping system for 16 SNP loci on mtDNA.
Dan WU ; Yan-Chai NIE ; Yu CAO ; Yu CAO ; Huai-Gu ZHOU
Journal of Forensic Medicine 2014;30(1):47-49
OBJECTIVE:
To establish a multiplex genotyping system of mtDNA SNP.
METHODS:
A multiplex analysis system of 16-plex mtDNA SNP loci was established with allele specific PCR and capillary electrophoresis genotyping technology. Fifty samples from unrelated Chinese Han individuals were typed with the multiplex system. The multiplex assay was validated by comparing with the direct sequencing method.
RESULTS:
The genotypes of all 50 samples were correctly determined by the multiplex system. The optimal genotypic graphs were obtained with an input DNA of 0.5-10 pg, and the typing results were completely consistent with those by direct sequencing method.
CONCLUSION
The established multiplex system by allele specific PCR has high sensitivity, operational simplicity and high accuracy. It provides an effective and high output method for mtDNA SNP typing.
Alleles
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DNA
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DNA, Mitochondrial
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Genotype
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Genotyping Techniques
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Humans
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Polymerase Chain Reaction/methods*
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Polymorphism, Single Nucleotide
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Sequence Analysis, DNA
9.Genetic polymorphisms of KIR2DS4 gene among ethnic Hans from southern China.
Jianxin ZHEN ; Guobin ZHANG ; Qiong YU ; Liumei HE ; Yunping XU ; Hongyan ZOU ; Zhihui DENG
Chinese Journal of Medical Genetics 2017;34(1):21-25
OBJECTIVETo study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.
METHODSGenomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.
RESULTSAmong all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.
CONCLUSIONThe present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; Haplotypes ; Humans ; Polymorphism, Genetic ; Receptors, KIR ; genetics ; Sequence Analysis, DNA ; methods
10.A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique.
Jun-feng WEI ; Tai-song ZHANG ; Hui-hong HUANG ; Yan-li ZENG ; Fan ZHANG ; Jun-jie WANG ; Bin ZHOU ; Ying-song WU ; Shu-wen LIU ; Jin-lin HOU ; Ya-bing GUO ; Yuan-ping ZHOU
Journal of Southern Medical University 2010;30(10):2270-2276
OBJECTIVETo develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.
METHODSThe primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.
RESULTSOf the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.
CONCLUSIONThis method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.
Genes, Viral ; Genotype ; Genotyping Techniques ; methods ; Hepacivirus ; classification ; genetics ; Hepatitis C ; virology ; Humans ; Immunoblotting ; Nucleic Acid Hybridization ; Reverse Transcriptase Polymerase Chain Reaction