Improved detection of global copy number variation using high density, non-polymorphic oligonucleotide probes

Goal: To identify copy number variation in normal individuals using high density, non-polymorphic oligonucleotide probes Background DNA sequence diversity within the human genome may be more greatly affected by copy number variations (CNVs) than single nucleotide polymorphisms (SNPs). Although the importance of CNVs in genome wide association studies (GWAS) is becoming widely accepted, the optimal methods for identifying these variants are still under evaluation. We have previously reported a comprehensive view of CNVs in the HapMap DNA collection using high density 500K EA (Early Access) SNP genotyping arrays which revealed greater than 1,000 CNVs ranging in size from 1kb to over 3Mb. Although the arrays used most commonly for GWAS predominantly interrogate SNPs, CNV identification and detection does not necessarily require the use of DNA probes centered on polymorphic nucleotides and may even be hindered by the dependence on a successful SNP genotyping assay. Results In this study, we have designed and evaluated a high density array predicated on the use of non-polymorphic oligonucleotide probes for CNV detection. This approach effectively uncouples copy number detection from SNP genotyping and thus has the potential to significantly improve probe coverage for genome-wide CNV identification. This array, in conjunction with PCR-based, complexity-reduced DNA target, queries over 1.3M independent NspI restriction enzyme fragments in the 200bp to 1100bp size range, which is a several fold increase in marker density as compared to the 500K EA array. In addition, a novel algorithm was developed and validated to extract CNV regions and boundaries. Conclusions Using a well-characterized pair of DNA samples, close to 200 CNVs were identified, of which nearly 50% appear novel yet were independently validated using quantitative PCR. The results indicate that non-polymorphic probes provide a robust approach for CNV identification, and the increasing precision of CNV boundary delineation should allow a more complete analysis of their genomic organization. Keywords: Copy number variation (CNV) detection A set of five genomic DNA samples containing different numbers of X chromosomes (1X to 5X sample set, including NA15510 and NA10851) were hybridized to Nsp copy number (CN) arrays in triplicate to evaluate detection of copy number variation using high density, non-polymorphic oligonucleotide probes. 6 Hapmap samples were hybridized to Nsp CN arrays to evaluate Mendelian inheritance of CNVs.

研究目标：利用高密度非多态性寡核苷酸探针，识别正常个体体内的拷贝数变异（Copy Number Variation, CNV）。

研究背景：人类基因组内的DNA序列多样性，受拷贝数变异（CNV）的影响可能远超单核苷酸多态性（Single Nucleotide Polymorphism, SNP）。尽管拷贝数变异在全基因组关联研究（Genome Wide Association Studies, GWAS）中的重要性已获广泛认可，但用于识别此类变异的最优方法仍处于评估阶段。本团队此前曾采用高密度500K EA（Early Access，早期获取）SNP基因分型芯片，对HapMap DNA样本集的拷贝数变异开展全面分析，共鉴定出超过1000个长度介于1kb至3Mb以上的拷贝数变异。尽管当前全基因组关联研究常用的芯片多以检测SNP为核心，但拷贝数变异的识别与检测未必需要依赖靶向多态性核苷酸的DNA探针，甚至可能因依赖成功的SNP基因分型实验而受到限制。

研究结果：本研究设计并评估了一款基于非多态性寡核苷酸探针的高密度芯片，用于拷贝数变异检测。该方法可将拷贝数检测与SNP基因分型有效解耦，因此具备显著提升全基因组拷贝数变异识别探针覆盖度的潜力。本芯片结合基于聚合酶链式反应（Polymerase Chain Reaction, PCR）的复杂度降低型DNA靶标，可对200bp至1100bp范围内的超过130万个独立NspI限制性内切酶片段进行检测，其标记密度较500K EA芯片提升数倍。此外，本研究还开发并验证了一款新型算法，用于提取拷贝数变异区域及其边界。

研究结论：借助一对经过充分表征的DNA样本，本研究共鉴定出近200个拷贝数变异，其中近50%为全新发现，并通过定量聚合酶链式反应（Quantitative PCR, qPCR）完成了独立验证。研究结果表明，非多态性探针可为拷贝数变异识别提供一种可靠的研究手段，而拷贝数变异边界界定精度的提升，将有助于更全面地解析其基因组组织特征。

关键词：拷贝数变异（CNV）检测

选取5组包含不同X染色体拷贝数的基因组DNA样本（1X至5X样本集，包含NA15510与NA10851），将其与Nsp拷贝数（CN）芯片进行三次重复杂交，以评估利用高密度非多态性寡核苷酸探针检测拷贝数变异的效果。选取6个HapMap样本与Nsp CN芯片进行杂交，以评估拷贝数变异的孟德尔遗传特征。