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

doi:10.11843/j.issn.0366-6964.2024.08.017

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3436-3445.doi: 10.11843/j.issn.0366-6964.2024.08.017

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

牛一凡¹^,²(), 李崇阳², 杨柏高², 张培培², 张航², 冯肖艺², 曹建华², 余洲², 马友记¹^,*(), 赵学明²^,*()

1. 甘肃农业大学动物科学技术学院，兰州 730070
2. 中国农业科学院北京畜牧兽医研究所，北京 100193

收稿日期:2023-12-11 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 马友记,赵学明 E-mail:nyf.niuyifan@qq.com;yjma@gsau.edu.cn;zhaoxueming@caas.cn
作者简介:牛一凡(1999-)，男，河北邯郸人，硕士生，主要从事动物繁殖研究，E-mail: nyf.niuyifan@qq.com
基金资助:
国家重点研发计划政府间重点专项(2022YFE0100200);国家自然科学基金国际合作项目(32161143032);农业农村部和财政部资助：现代农业产业技术体系资助(CARS-36);国家家养动物种质资源库、中国农业科学院科技创新工程(ASTIP-IAS06)

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

Yifan NIU¹^,²(), Chongyang LI², Baigao YANG², Peipei ZHANG², Hang ZHANG², Xiaoyi FENG², Jianhua CAO², Zhou YU², Youji MA¹^,*(), Xueming ZHAO²^,*()

1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2023-12-11 Online:2024-08-23 Published:2024-08-28
Contact: Youji MA, Xueming ZHAO E-mail:nyf.niuyifan@qq.com;yjma@gsau.edu.cn;zhaoxueming@caas.cn

摘要/Abstract

摘要：

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。

关键词: 全基因组扩增, MDA, MALBAC, 微量DNA扩增体系

Abstract:

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 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 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.

Key words: whole genome amplification, MDA, MALBAC, trace DNA amplification system

中图分类号:

S823.2

牛一凡, 李崇阳, 杨柏高, 张培培, 张航, 冯肖艺, 曹建华, 余洲, 马友记, 赵学明. 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[J]. 畜牧兽医学报, 2024, 55(8): 3436-3445.

Yifan NIU, Chongyang LI, Baigao YANG, Peipei ZHANG, Hang ZHANG, Xiaoyi FENG, Jianhua CAO, Zhou YU, Youji MA, Xueming ZHAO. Evaluation of the Effect of Different Single Cell Whole Genome Amplification Systems on the Amplification of Bovine Trace Blood DNA[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3436-3445.

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样品名称 Sample	浓度/(ng·μL^-1) Concentration	体积/μL Volume	总质量/μg Total mass
A	49.9	45	2.25
B	35.8	45	1.61
C	65.5	45	2.95

体系名称 System name	扩增产物浓度/(ng·μL^-1) Post-amplification concentration	体积/μL Volume	扩增产物总质量/μg Total mass	扩增倍数 Amplification multiple
MDA	138.67±14.43^A	50	6.93±0.72^A	6.1×10³~7.35×10^{3 A}
MALBAC	19.63±0.98^B	65	1.27±0.06^B	1.2×10³~1.31×10^{3 B}

样本名称 Sample name	原始数据/G Raw data	原始reads Raw reads	过滤后的碱基量/G Clean base	过滤后的reads数目 Clean reads	有效碱基比率/% Effective rate	Q20/%	Q30/%	GC/%
NC-A	68.75	229 182 528	68.75	229 028 205	99.93	97.29	92.28	42.72
NC-B	72.08	240 265 726	71.98	240 132 402	99.94	97.28	92.27	42.77
NC-C	65.32	217 735 046	65.21	217 602 659	99.94	97.21	92.07	43.15
MDA-A	86.45	288 158 236	109.88	366 596 150	99.94	96.56	90.30	41.88
MDA-B	111.18	370 586 573	60.65	202 316 763	99.94	97.81	93.61	41.79
MDA-C	103.68	345 605 125	73.80	246 140 648	99.94	97.40	92.58	41.69
MALBAC-A	110.05	366 826 265	86.31	287 837 233	99.89	96.65	90.58	42.69
MALBAC-B	60.73	202 436 895	110.98	370 296 021	99.92	97.13	91.85	42.85
MALBAC-C	73.89	246 290 038	103.49	345 336 461	99.92	96.93	91.32	43.16
小计Total	752.13	2 507 086 432	751.05	2 505 286 542
总体均值Average					99.93	97.14	91.87	42.52
Average

样本名称 Sample name	总reads数目 Total reads	比对reads数目 Mapped reads	比对率/% Mapped ratio	正确比对reads数目 Properly mapped reads	正确比对率/% Properly mapped ratio	重复率/% Dup ratio
NC-A	458 957 384	458 409 872	99.88	450 607 404	98.37	2.27
NC-B	481 227 738	480 697 245	99.89	472 836 054	98.45	2.15
NC-C	436 169 391	435 532 805	99.85	428 305 230	98.41	2.33
MDA-A	589 036 553	588 273 032	99.87	539 900 540	93.79	1.34
MDA-B	755 359 505	754 544 244	99.89	711 784 484	96.11	1.55
MDA-C	707 734 945	706 809 010	99.87	655 106 940	94.85	1.56
MALBAC-A	757 009 041	755 925 119	99.86	689 538 526	94.05	2.37
MALBAC-B	418 273 461	417 698 203	99.86	379 232 754	93.72	1.50
MALBAC-C	507 852 724	507 177 062	99.87	463 366 622	94.13	2.11
小计Total	5 111 620 742	5 105 066 592		4 790 678 554
总体均值Average			99.87		95.76	1.91
Average

对照组 Control group	扩增组 Amplification group	分型一致率/% Consistent rate of typing	分型一致位点数 Common loci	分型不一致位点数 Differed loci	检出位点数 Call loci	对照组缺失的位点 Control group drop loci
NC-A	MDA-A	97.91	11 352 029	242 889	11 594 918	37 974
	MALBAC-A	88.93	9 415 570	1 172 572	10 588 142	25 968
NC-B	MDA-B	98.07	11 404 499	223 896	11 628 395	33 366
	MALBAC-B	86.79	8 579 289	1 305 443	9 884 732	15 952
NC-C	MDA-C	97.89	11 391 983	245 867	11 637 850	39 855
	MALBAC-C	86.79	8 750 915	1 331 445	10 082 360	19 658

对照组 Control group	扩增组 Amplification group	扩增组缺失位点 Amplification group drop loci	两组同时缺失位点 Simultaneous drop loci in both groups	等位基因缺失率/% Allele drop rate	假阳性率/% False positive rate	检出率/% Call rate
NC-A	MDA-A	78 636	79 601	0.93	0.81	98.66
	MALBAC-A	1 085 412	91 607	4.88	4.46	90.02
NC-B	MDA-B	66 664	62 704	0.83	0.79	98.90
	MALBAC-B	1 810 327	80 118	7.19	3.67	83.97
NC-C	MDA-C	53 837	59 587	0.79	1.00	99.04
	MALBAC-C	1 609 327	79 784	6.81	4.14	85.67

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

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