不同单细胞全基因组扩增体系扩增牛微量血液DNA效果评价

Evaluation of the Effect of Different Single Cell Whole Genome Amplification Systems on the Amplification of Bovine Trace Blood DNA

旨在通过二代测序技术评估不同单细胞全基因组扩增(single cell whole genome amplification,scWGA)体系对牛微量血液基因组DNA的扩增效果,建立微量DNA全基因组扩增体系。本研究分别采用MDA(multiple displacement amplification)和MALBAC(multiple annealing and looping-based amplification cycles)两种scWGA体系对华西牛1 ng血液基因组DNA进行全基因组扩增,随后基于两种体系的扩增产物以及原始未稀释血液DNA构建测序文库,利用DNBSEQ-T7RS测序平台进行全基因组测序(whole genome sequencing,WGS),通过比较扩增产物片段大小、浓度、总质量评估扩增效率,通过分析GC含量、测序覆盖度、基因分型一致率、基因型检出率等评估两种体系的扩增效果。结果显示,MDA体系的扩增产物片段大于MALBAC体系(8 kb vs.0.2~2 kb),产物浓度和总质量显著高于MALBAC体系(P<0.05)。基于测序数据,1×和5×测序深度下,MDA体系的基因组覆盖度显著高于MALBAC体系(P<0.05),此外,MDA体系的分型一致率、检出率显著高于MALBAC体系,而等位基因缺失率、假阳性率显著低于MALBAC体系(P<0.05)。综上,本研究揭示了MDA和MALBAC两种扩增体系基于华西牛1 ng血液基因组DNA扩增的体系特点,为改进现有华西牛胚胎基因组选择中的关键扩增技术提供理论依据,促进华西牛遗传育种进展。

The purpose of this paper was to evaluate the amplification effect of different single cell whole genome amplification(scWGA)systems on bovine trace blood genomic DNA by next-generation sequencing,and to establish a trace DNA whole genome amplification system.In this study,the whole genome of 1 ng blood genomic DNA of Huaxi cattle was amplified by MDA(multiple displacement amplification)and MALBAC(multiple annealing and looping-based amplification cycles)scWGA systems,and then the sequencing library was constructed based on the amplification products of the two systems and the original undiluted blood DNA.The whole genome sequencing(WGS)was used performed using the DNBSEQ-T7RS sequencing platform.The amplification efficiency was evaluated by comparing the fragment′s size,concentration and total mass of the amplified products.The amplification effects of the two systems were evaluated by analyzing GC content,average depth,consistent rate of typing and call rate.The results showed that the fragment of the amplified product in MDA system was larger than that in MALBAC system(8 kb vs.0.2-2 kb),the concentration and total mass of the product were significantly higher than those in MALBAC system(P<0.05).Based on the sequencing data,the genome coverage of MDA system was significantly higher than that of MALBAC system at 1×and 5×sequencing depth(P<0.05).In addition,the consistency of typing rate and call rate of MDA system were significantly higher than MALBAC system,while the allele drop rate and false positive rate of MALBAC system were significantly lower than MALBAC system(P<0.05).In summary,this study revealed that the characteristics of two amplification systems of MDA and MALBAC were based on the characteristics of DNA amplification of Huaxi cattle 1 ng blood genome,which provided a theoretical basis for improving the key amplification techniques in Huaxi cattle embryo genome selection and promoting the progress of Huaxi cattle genetics and breeding.