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

Abstract

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

CLC Number:

S828.2

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.

Figures/Tables 17

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References 33

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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

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

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细胞数 Cell number	项目 Item	TIANamp Micro DNA Kit	MagicPure Microbiome DNA Isolation Kit	REPLI-g^® Single Cell Kit
3	检出的SNP数量Number of detected SNPs	11 928	1 456 232	10 496 024
	占dbSNP库比例/% Percentage of dbSNP	49.86	73.34	82.40
5	检出的SNP数量Number of detected SNPs	50 271	329 523	11 264 639
	占dbSNP库比例/% Percentage of dbSNP	61.91	68.21	81.94
7	检出的SNP数量Number of detected SNPs	51 742	1 547 788	11 190 573
	占dbSNP库比例/% Percentage of dbSNP	47.56	70.55	82.40
10	检出的SNP数量Number of detected SNPs	80 375	2 050 007	10 387 060
	占dbSNP库比例/% Percentage of dbSNP	54.86	72.69	82.46

细胞数 Cell number	检出SNPs数量 Number of detected SNPs	检出INDELs数量 Number of detected INDELs	占dbSNP库比例/% Percentage of dbSNP
5	2 670 902±1 639 522	418 698±253 237	87.98±1.93
7	3 976 749±414 046	628 196±52 588	89.26±0.93

细胞数 Cell number	样品名 Sample name	检出SNPs位点数 Number of detected SNPs	检出率/% Call rate	平均检出率/% Mean call rate
5	5-1	73 319	76.97	74.09±12.08
	5-2	57 940	60.83
	5-3	80 475	84.48
7	7-1	68 707	72.13	81.52±8.19
	7-2	83 092	87.23
	7-3	81 151	85.19

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