Qi Yuan-Yuan, Zhou Xu-Jie, Bu Ding-Fang, Hou Ping, Lv Ji-Cheng, Zhang Hong
Renal Division, Peking University First HospitalBeijing, China; Peking University Institute of NephrologyBeijing, China; Key Laboratory of Renal Disease, Ministry of Health of ChinaBeijing, China; Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of EducationBeijing, China.
Research Central Institute, Peking University First Hospital Beijing, China.
Front Genet. 2016 Dec 26;7:220. doi: 10.3389/fgene.2016.00220. eCollection 2016.
Low copy numbers (CNs) has been associated with susceptibility to several systemic autoimmune diseases. However, inconsistent associations were reported and errors caused by shaky methods were suggested to be the major causes. In large scale case control association studies, robust copy number determination method is thus warranted, which was the main focus of the current study. In the present study, CNs of 90 HapMap Asians were firstly checked using four assays including paralog ratio test combined with restriction enzyme digest variant ratio (PRT-REDVR), real-time quantitative (qPCR) using TaqMan assay, real-time qPCR using SYBR Green dye and short tenden repeat (STR). To improve the comparison precision reproductively, the results were compared with those from recently released sequencing data from 1000 genomes project as well as whole-genome tiling BAC array data. The tendencies of inconsistent samples by these methods were also characterized. Refined in-home TaqMan qPCR assay showed the highest correlation with array-CGH results ( = 0.726, < 0.001) and the highest concordant rate with 1000 genome sequencing data ( 91.76%, 85.88%, and 81.18%). For samples with copy number variations, comprehensive analysis of multiple methods was required in order to improve detection accuracy. All these method were prone to detect copy number to be higher than that from direct sequencing. All the four PCR based CN determination methods (qPCR using TaqMan probes or SYBR Green, PRT, STR) were prone to higher estimation errors and thus may lead to artificial associations in large-scale case-control association studies. But different to previous reports, we observed that properly refined TaqMan qPCR assay was not inferior to or even more accurate than PRT when using sequencing data as the reference.
低拷贝数（CNs）已被证明与多种系统性自身免疫性疾病的易感性相关。然而，此前报道的相关性并不一致，且有人认为方法不可靠所导致的误差是主要原因。因此，在大规模病例对照关联研究中，需要一种可靠的拷贝数测定方法，这也是本研究的主要关注点。在本研究中，首先使用四种检测方法对90名HapMap亚洲人的拷贝数进行检测，包括旁系同源比率测试结合限制性内切酶消化变异比率（PRT-REDVR）、使用TaqMan检测法的实时定量（qPCR）、使用SYBR Green染料的实时qPCR以及短串联重复序列（STR）。为了提高结果比较的重复性和准确性，将这些结果与最近发布的千人基因组计划测序数据以及全基因组平铺BAC阵列数据进行了比较。同时，还对这些方法检测结果不一致的样本的趋势进行了分析。优化后的家用TaqMan qPCR检测法与阵列比较基因组杂交（array-CGH）结果的相关性最高（r = 0.726，P < 0.001），与千人基因组测序数据的一致性率也最高（分别为91.76%、85.88%和81.18%）。对于存在拷贝数变异的样本，需要综合多种方法进行分析，以提高检测准确性。所有这些方法都倾向于检测到比直接测序更高的拷贝数。基于PCR的所有四种拷贝数测定方法（使用TaqMan探针或SYBR Green的qPCR、PRT、STR）都容易产生较高的估计误差，因此可能会在大规模病例对照关联研究中导致人为关联。但与之前的报道不同，我们观察到，以测序数据为参考时，经过适当优化的TaqMan qPCR检测法并不比PRT差，甚至更准确。
