基于不同方法的微量细胞全基因组遗传变异检测和比较分析

Detection and Comparative Analysis of Genomic Genetic Variations in Trace Cells Using Different Methods

旨在用不同方法对微量细胞全基因组遗传变异进行检测和比较分析。本研究首先以3、5、7、10个猪耳缘成纤维细胞为试验材料,利用不同方法提取微量细胞基因组,进行全基因组测序(whole genome sequencing,WGS),比较不同细胞数及不同提取方法之间的遗传变异检测性能。随后利用牛囊胚的5个和7个滋养外胚层细胞获得的基因组进行全基因组测序和SNP芯片技术的比较分析,每组均进行3次重复。结果表明,针对不同细胞数,基于全基因组扩增技术(whole genomic amplification,WGA)的MDA(multiple displacement amplification)方法均比其他方法的DNA产物浓度、测序质量及SNP检出性能效果更优。使用REPLI-g^((R)) Single Cell Kit扩增7、10细胞数的DNA浓度显著高于3、5细胞数,但质量评估的各项性能基本无显著差异,且不同细胞数检测到的SNP位点数量相似。5、7细胞数的Illumina Bovine GGP芯片SNP位点call rate分别为74.09%和81.52%。全基因组测序相较于芯片可以获得更丰富的遗传变异信息,但两种检测手段共同检测到的SNP以及基因型相同的位点数占比较低。综上所述,本研究系统比较了不同细胞数下不同微量细胞基因组提取方法以及不同全基因组遗传变异检测技术的各项性能,结果显示利用REPLI-g^((R)) Single Cell Kit扩增7细胞进行二代测序可得到较为准确且稳定的结果,本研究建立了一套较为可靠的家畜胚胎微量细胞样品基因组提取和遗传变异检测方案,为将来实现准确的胚胎基因组选择和胚胎质量评估具有重要的意义和价值。

The aim of this study was to detect and compare the genetic variation of the whole genome of trace cells by different methods.In this study,3,5,7 and 10 cell numbers from pig ear fibroblasts were used as experimental materials,and trace cell genomes were extracted by different methods for whole genome sequencing(WGS)to compare the performance of genetic variation detection among different cell numbers and extraction methods.Subsequently,comparative anal-ysis of whole genome sequencing and SNP chip technology was conducted using the genomes ob-tained from 5 or 7 bovine trophectoderm cells,with 3 replicates per group.The results showed that the multiple displacement amplification(MDA)method based on whole genomic amplifica-tion(WGA)technology had better DNA product concentration,sequencing data quality and SNP detection performance than other methods for different cell numbers.The DNA concentrations of 7 and 10 cell numbers amplified by REPLI-g^((R)) Single Cell Kit were significantly higher than those of 3 and 5 cell numbers,but there was no significant difference in the performance of quality as-sessment,and the number of SNPs detected was similar in different cell numbers.The call rates of Illumina Bovine GGP chip with 5 and 7 cell numbers were 74.09%and 81.52%,respectively.The results showed that more genetic variation information could be obtained by whole genome sequencing compared to SNP chips,but the proportion of SNPs and the number of loci with the same genotype detected by both methods was low.In conclusion,this study systematically com-pared the performance of genome extraction methods and whole genome genetic variation detec-tion techniques for different cell numbers.The results showed that NGS amplification of 7 cells using the REPLI-g^((R)) Single Cell Kit could obtain relatively accurate and stable results.This study established a reliable genome extraction and genetic variation detection scheme for trace cell sam-ples of livestock embryos,which is of important significance and value for accurate embryo ge-nomic selection and embryo quality assessment in the future.