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目的探讨NanoDrop检测D_(260)/D_(230)值对全基因组关联研究(GWAS)中DNA标本质检的意义。方法收集1 494例强直性脊柱炎(AS)患者的血液DNA样本,分别用NanoDrop和PicoGreen检测样本浓度。第一阶段,对24例NanoDrop和PicoGreen浓度>50ng/μL的DNA样本行Omni中华8芯片检测,比较16例芯片成功样本与8例失败样本的D_(260)/D_(280)值及D_(260)/D_(230)值。第二阶段,选取1 122例NanoDrop和PicoGreen检测浓度均大于50ng/μL DNA样本行管家基因GAPDH的PCR检测,并对PCR反应成功的样本行Omni中华8芯片检测。采用Mann-Whitney U检验比较PCR反应成功与失败DNA样本的D_(260)/D_(230)值,采用受试者工作特征(ROC)曲线评价D_(260)/D_(230)值对PCR结果的鉴别效率。结果第一阶段中,芯片成功与失败样本的D_(260)/D_(280)值差异无统计学意义(P=0.444),而D_(260)/D_(230)值差异有统计学意义(Z=-3.920,P<0.001);第二阶段中,PCR成功的DNA样本基因分型检测成功率为100%,PCR成功与失败组的D_(260)/D_(230)值比较差异有统计学意义(Z=-5.983,P<0.01)。D_(260)/D_(230)值预测PCR结果的曲线下面积(AUC)为0.727;最佳诊断点的D_(260)/D_(230)值为0.89;特异度为0.95时的D_(260)/D_(230)值为2.305。结论在进行GWAS时,浓度及D_(260)/D_(280)值均较好而D_(260)/D_(230)值较低的DNA样本可能含有较多杂质,需联用PCR检测以确保样本质量;D_(260)/D_(230)值≥2.305时,样本纯度满足GWAS芯片检测的要求,可省略PCR检测。
Objective To investigate the significance of NanoDrop detection of D 260 (260) / D 230 values in DNA sequencing in genome-wide association study (GWAS). Methods Blood DNA samples from 1 494 patients with ankylosing spondylitis (AS) were collected and the concentrations of the samples were determined with NanoDrop and PicoGreen respectively. In the first phase, 24 cases of NanoDrop and PicoGreen DNA samples with concentration> 50ng / μL were tested by Omni Zhonghua 8 chip. The D 260 / D 280 values and D_ 260) / D_ (230) value. In the second stage, 1 122 cases of NanoDrop and PicoGreen were selected for PCR detection of housekeeping gene GAPDH with the concentration higher than 50ng / μL DNA samples, and Omni Zhonghua 8 chip was selected for the successful PCR reaction. The D_ (260) / D_ (230) values of DNA samples were compared by Mann-Whitney U test, and the PCR results of D_ (260) / D_ (230) were evaluated by receiver operating characteristic Identification efficiency. Results There was no significant difference in D_ (260) / D 280 between the successful and the failed samples in the first stage (P = 0.444), while the value of D_ (260) / D_ (230) was statistically significant ( Z = -3.920, P <0.001). In the second stage, the success rate of genotyping PCR was 100%, and the differences of D 260 / D 230 between PCR success and failure were statistically significant Significance (Z = -5.983, P <0.01). The area under the curve (AUC) of the predicted value of D 260 / D 230 was 0.727, the value of D 260 / D 230 of the best diagnostic point was 0.89, and the value of D 260 for the specificity of 0.95 ) / D_ (230) value is 2.305. Conclusions When the GWAS is performed, DNA samples with better D 260 / D 280 values and lower D 260 / D 230 values may contain more impurities and PCR tests should be performed to ensure that Sample quality; D_ (260) / D_ (230) value ≥2.305, sample purity to meet the requirements of GWAS chip detection, PCR detection can be omitted.