质粒连接克隆法用于提高单细胞全基因组扩增产物覆盖率及质量的初探

The analysis of plasmid-cloning method to improve the coverage and quality of single cell whole genome amplification products

目的:评估利用质粒连接克隆技术提高稀有细胞全基因组扩增(WGA)产物覆盖率及质量的可行性。方法:以54例宫颈脱落滋养层细胞和循环肿瘤细胞的WGA产物为研究对象,利用质粒连接克隆技术,通过质粒载体pMDTM19-T将单细胞WGA产物转入大肠杆菌DH5α感受态细胞进行增殖获取WGA产物的克隆产物。设计22条常染色体中22个特定位点引物进行扩增,根据扩增阳性率计算单细胞WGA产物克隆前后的覆盖率,并利用配对t检验及散点图进行统计学分析。随机选取样本,利用Sanger测序和短串联重复序列技术进行产物质量验证。结果:克隆后单细胞WGA产物的覆盖率有所提高(克隆前73%、克隆后79%),尤其对宫颈脱落滋养层细胞效果更为显著(克隆前72%、克隆后81%);在随机选取的12例样本中,克隆后样本的Sanger测序峰图质量和成功率均优于克隆前;此外,随机选取的2例克隆后的样本检测出的STR基因座均显著多于克隆前的。结论:质粒连接克隆法在一定程度上能提高单细胞WGA产物覆盖率及质量,有助于更深入地研究稀有细胞的生物学意义及临床价值。

Objective:To evaluate the feasibility of using plasmid-cloning to improve the coverage and quality of WGA products from rare cells.Methods:In this study,54 cases of WGA products from trophoblast cells and circulating tumour cells were investigated.Using plasmid-cloning technology,the WGA products of single cells were transferred into Escherichia coli DH5αcompetent cells by plasmid vector pMDTM19-T for prolifertion.Primers of 22 specific loci in 22 autosomes were designed for amplification,and the coverage of single-cell WGA products before and after cloning was calculated according to the positive rate of amplification.Then the paired t-test and scatter plot were used for statistical analysis.Sanger sequencing and short tandem repeat technology were used to verify the product quality in the randomly selected samples.Results:After cloning,the mean coverage of single-cell WGA products was improved(pre-clone 73%,post-clone 79%),especially for trophoblast cells(pre-clone 72%,post-clone 81%).Among the 12 randomly selected samples,the peak map quality and success rate of Sanger sequencing of the cloned samples were better than those before cloning.In addition,the number of detected STR loci in the cloned samples was significantly higher than those before cloning in the two randomly selected samples.Conclusion:The plasmid-cloning method can improve the coverage and quality of single-cell WGA products to a certain extent.This strategy may helpful to further exploring the biological significance and clinical value of rare cells.