Evaluation of the amplicons for detection of known CNVs using Cs22 HR-CGH

Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above. Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH) The overall goal of this part of study was a validation of the quality of the amplicons from different amounts of original starting gDNA, good quality or partially degraded gDNA, with our new procedure Wpa, or our intermediate testing procedure Wpa-40C, with native gDNA as control, in terms of the detectability of the known large-scale heretozygous deletions and duplications (CNVs), or small-scale (sub-kb) CNVs. Amplified or native genomic DNA isolated from patients with known CNVs (Cy3) was co-hybridized with in-parallel amplified or native reference gDNA pool (Promega; Cy5).

对复杂DNA池实现高特异性扩增且无偏倚或模板非依赖产物(Template-independent products, TIPs)，仍是一项挑战性难题。本研究开发了一种采用phi29 DNA聚合酶(phi29 DNA polymerase)与海藻糖，并优化扩增调控流程的方法，可从低至亚飞克级的DNA起始量中，制备出微克级特异性扩增产物，且无TIPs生成。针对原本质量优异的基因组DNA(genomic DNA, gDNA，样本编号05-050)或部分降解的gDNA（样本编号04-018），分别以5 ng和0.5 ng作为起始量所获得的扩增产物，通过高分辨率比较基因组杂交(High resolution comparative genome hybridization, HR-CGH)，可忠实检出22号染色体上所有此前已知的杂合性片段重复与缺失（长度范围3 Mb至18 kb），甚至可精准检测到小于1 kb的纯合缺失。具体而言，以5 ng起始gDNA的扩增产物进行HR-CGH检测，可在两名不同先证者的样本中检出所有此前已知的22号染色体片段畸变，其检测结果与同一先证者的天然gDNA的HR-CGH结果无显著差异（图4、补充图S3及S4）。断裂位点亦可被精准定位，其中包括样本05-050中的杂合性基因组片段重复（3个拷贝，长度3 Mb，图4），以及样本04-018中的两种不同杂合性缺失（1个拷贝，分别为1.4 Mb和18 kb，图S4）；所有上述变异均位于低拷贝重复序列(Low copy repeats, LCRs)区域内或其侧翼区域。此外，此前已知的、存在于样本04-018与05-050中的975 bp纯合缺失，也被准确检出（05-050的检测结果见图4c）；不过在部分样本（如04-018）中，其信号噪声略高于未扩增的DNA（补充图S4d）。与之形成鲜明对比的是，采用Wpa-40℃扩增法会产生大量信号噪声，无法成功检出上述拷贝数变异（图4、补充图S3及S4）。值得注意的是，采用Wpa法以0.5 ng起始gDNA的扩增产物进行HR-CGH检测，虽信号噪声更高，但仍可清晰检出已知的拷贝数变异(Copy Number Variations, CNVs)（补充图S4）。以0.1 ng起始gDNA的扩增产物因信号变异性较高，无法明确显示CNVs，但CNV的整体模式大多得以保留（样本04-018的结果见补充图S4）。本研究同时也注意到扩增产物中存在一定的位点失衡现象，但该现象可被有效抑制；当起始DNA量高于特定阈值时，该现象具有良好的可重复性，且如前文所述，若平行使用同一批扩增的参考样本，即可有效补偿该失衡。关键词：全池扩增(Whole-pool amplification, Wpa)、高分辨率比较基因组杂交(High resolution comparative genome hybridization, HR-CGH)。本部分研究的整体目标为：以天然gDNA作为对照，评估本研究开发的新型Wpa法与中间测试方法Wpa-40℃扩增法，对不同起始量、不同质量（良好或部分降解）的原始gDNA所制备的扩增产物的质量，重点考察其对已知大型杂合性缺失与重复（拷贝数变异，CNVs）或小型（亚kb级）CNVs的检测能力。将携带已知CNVs的患者来源的扩增或天然基因组DNA（标记为Cy3），与平行扩增或天然的参考gDNA池（普洛麦格(Promega)，标记为Cy5）进行共杂交。