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

doi:10.11843/j.issn.0366-6964.2024.10.007

摘要/Abstract

摘要：

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^® 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^® 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。

关键词: 微量细胞, 胚胎, 全基因组扩增, 重测序, SNP芯片, 遗传变异

Abstract:

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 analysis of whole genome sequencing and SNP chip technology was conducted using the genomes obtained 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 amplification (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^® 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 assessment, 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 compared the performance of genome extraction methods and whole genome genetic variation detection techniques for different cell numbers. The results showed that NGS amplification of 7 cells using the REPLI-g^® 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 samples of livestock embryos, which is of important significance and value for accurate embryo genomic selection and embryo quality assessment in the future.

Key words: trace cells, embryo, whole genome amplification, resequencing, SNP chip, genetic variation

中图分类号:

S828.2

石庆珍, 徐鸿洋, 张燕, 张毅, 王雅春, 韩建永, 姜力. 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[J]. 畜牧兽医学报, 2024, 55(10): 4311-4324.

Qingzhen SHI, Hongyang XU, Yan ZHANG, Yi ZHANG, Yachun WANG, Jianyong HAN, Li JIANG. Detection and Comparative Analysis of Genomic Genetic Variations in Trace Cells Using Different Methods[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4311-4324.

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细胞数 Cell number	EasyPure Micro Genomic DNA Kit	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	MALBAC^® Single Cell WGA Kit	GenomePlex^® Whole Genome Amplification	REPLI-g^® Single Cell Kit
7	0.05±0.06de	0.25±0.03d	0.04±0.03e	14.03±0.40b	8.86±0.87c	747.33±25.17a
10	0.01±0.00b	0.24±0.05b	0.12±0.03b	17.07±5.68b	26.33±6.71b	800.67±79.41a

项目 Item	EasyPure Micro Genomic DNA Kit	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	MALBAC^® Single Cell WGA Kit	GenomePlex^® Whole Genome Amplification	REPLI-g^® Single Cell Kit
原始碱基数/Gb Raw bases	5.57±1.94	6.60±9.94	5.01±6.65b	7.83±3.86	7.10±1.54	8.96±1.10a
过滤后碱基/Gb Clean bases	5.55±1.93	6.00±9.27	4.79±5.83b	7.43±2.50	7.00±1.52	8.87±1.08a
过滤后比例/% Clean rate	99.58±0.15	91.04±3.95	95.56±1.19	94.93±1.92	98.56±0.31	98.95±0.27
Q20/%	96.35±0.17a	96.64±0.13a	96.84±0.24a	96.89±0.06a	94.43±0.96b	96.89±0.31a
GC/%	46.58±0.84abc	48.20±5.68	41.95±1.12	46.30±0.59b	41.49±0.28c	40.08±0.26d

项目 Item	EasyPure Micro Genomic DNA Kit	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	MALBAC^® Single Cell WGA Kit	GenomePlex^® Whole Genome Amplification	REPLI-g^® Single Cell Kit
原始碱基数/Gb Raw bases	6.60±3.50	6.35±1.47	5.76±6.46	9.19±1.36	8.13±1.57	7.71±1.09
过滤后碱基/Gb Clean bases	6.54±3.30	5.66±1.46	5.47±6.25	8.44±1.37	7.96±1.52	7.64±1.08
过滤后比例/% Clean rate	99.12±0.48a	88.87±3.93	94.92±0.26b	91.63±2.30	97.92±0.28a	99.07±0.16a
Q20/%	95.92±1.01ab	96.53±0.23a	96.88±0.19a	97.03±0.17a	94.30±0.12b	97.01±0.04a
GC/%	41.45±0.10bc	48.78±1.54	42.55±0.22b	46.44±0.13a	41.35±0.07bc	39.95±0.32c

项目 Item	EasyPure Micro Genomic DNA Kit	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	MALBAC^® Single Cell WGA Kit	GenomePlex^® Whole Genome Amplification	REPLI-g^® Single Cell Kit
平均深度/X Mean	1.45±0.78bc	0.58±0.41c	1.45±0.62bc	2.78±0.08ab	2.53±0.55ab	3.53±0.44a
覆盖度/% Coverage	38.33±18.73	2.03±0.95d	35.87±14.19	51.87±1.22b	35.10±1.10c	83.53±1.75a
比对率/% Mapped rate	68.97±13.59	38.51±16.15	78.43±24.07	98.19±0.16	97.44±0.49	98.81±0.14
双端比对率/% Pair mapped rate	62.34±14.20	32.51±14.22	74.40±24.00	84.99±1.74	86.94±1.63	88.22±0.26

项目 Item	EasyPure Micro Genomic DNA Kit	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	MALBAC^® Single Cell WGA Kit	GenomePlex^® Whole Genome Amplification	REPLI-g^® Single Cell Kit
平均深度/X Mean	1.33±0.51b	1.04±0.11c	1.96±0.26	3.14±0.51a	2.85±0.56a	3.04±0.43a
覆盖度/% Coverage	14.83±5.00c	2.37±0.47c	43.20±12.55	51.67±1.88b	54.90±4.67b	80.23±4.50a
比对率/% Mapped rate	58.00±14.12	56.13±11.63	90.90±1.90	98.03±0.58	97.77±0.17b	98.84±0.09a
双端比对率/% Pair mapped rate	50.74±14.83	50.99±11.07	86.74±2.08	85.11±0.59b	84.45±1.06	88.17±0.42a