质粒DNA Fast NGS测序方法的开发和应用

doi:10.19586/j.2095-2341.2024.0039

生物技术进展 ›› 2024, Vol. 14 ›› Issue (4): 594-600.DOI: 10.19586/j.2095-2341.2024.0039

• 研究论文 • 上一篇

质粒DNA Fast NGS测序方法的开发和应用

宋辉¹^,²(), 曹文刚¹^,², 肖晓文¹^,², 杜军¹()

^1.北京擎科生物科技股份有限公司研究院，北京 100000
^2.湖北擎科生物科技有限公司研发部，湖北鄂州 431800

收稿日期:2024-03-01 接受日期:2024-05-20 出版日期:2024-07-25 发布日期:2024-08-07
通讯作者: 杜军
作者简介:宋辉 E-mail: songhui@tsingke.com.cn；
基金资助:
鄂州市科学技术局2022年科技计划项目-重点研发专项

Development and Application of Fast NGS Sequencing Method for Plasmid DNA

Hui SONG¹^,²(), Wengang CAO¹^,², Xiaowen XIAO¹^,², Jun DU¹()

^1.Research Institute，Beijing Tsingke Biotech Co. ，Ltd. ，Beijing 100000，China
^2.Research and Development Department，Hubei Tsingke Biotechnoloy Co. ，Ltd. ，Hubei Ezhou 431800，China

Received:2024-03-01 Accepted:2024-05-20 Online:2024-07-25 Published:2024-08-07
Contact: Jun DU

摘要/Abstract

摘要：

质粒DNA是最常用的基因运载工具，在基因合成技术中扮演着至关重要的角色。如何实现合成质粒DNA的准确且快速检测，是确保基因组完整性和提高基因合成效率的关键。尽管基于一代DNA的测序方法，其准确性已成为行业标准，但在检测通量、检测速度和检测成本等方面仍然存在局限性，这促使科学家们不断寻求新的解决方案。基于生物酶库，开发了DNA建库酶TN5，建立了高通量质粒DNA检测方案——Fast NGS。利用不同长度、不同质量的质粒DNA样本评估了Fast NGS的可行性，并对质粒DNA样本进行了高通量测序，最后对比了Fast NGS与Sanger测序的效率。结果表明，DNA建库酶TN5蛋白的纯度和质量符合二代测序要求。Fast NGS适用于3~8 kb基因合成质粒的测序检测，其检测通量高达2 500个·12 h^-1，测序成功率超过95%，测序准确性与一代测序相当，并且无明显序列偏好性。Fast NGS实现了质粒DNA的高通量、快速且低成本检测，为基因合成技术的发展提供了新的方向。

关键词: Fast NGS, 质粒DNA, 高通量测序, TN5

Abstract:

Plasmid DNA serves as the most commonly used gene delivery vehicle， playing a crucial role in gene synthesis technology. Achieving accurate and rapid detection of synthesized plasmid DNA is the key to ensuring the integrity of the genome and improving the efficiency of gene synthesis. DNA detection methods based on first-generation sequencing have established their accuracy as an industry standard， but they have limitations in terms of detection throughput， speed， and cost， which has prompted scientists to continuously seek new solutions. Based on the biological enzyme library， the DNA library construction enzyme TN5 was developed， and a high-throughput plasmid DNA detection solution， named Fast NGS， was established. The feasibility of Fast NGS was evaluated using plasmid DNA samples of different lengths and qualities and high-throughput sequencing of plasmid DNA samples was performed. Finally， the efficiency of Fast NGS and Sanger sequencing was compared. The results showed that the purity and quality of the DNA library construction enzyme TN5 protein met the requirements of next-generation sequencing. Fast NGS is suitable for sequencing of 3~8 kb gene synthesis plasmids， and its detection throughput is up to 2 500 per 12 h， with a sequencing success rate of over 95%. The sequencing accuracy is comparable to that of first-generation sequencing， and there is no significant sequence preference. Fast NGS achieves high-throughput， rapid， and low-cost detection of plasmid DNA， providing a new direction for the development of gene synthesis technology.

Key words: Fast NGS, plasmid DNA, high-throughput sequencing, TN5

中图分类号:

Q819

宋辉, 曹文刚, 肖晓文, 杜军. 质粒DNA Fast NGS测序方法的开发和应用[J]. 生物技术进展, 2024, 14(4): 594-600.

Hui SONG, Wengang CAO, Xiaowen XIAO, Jun DU. Development and Application of Fast NGS Sequencing Method for Plasmid DNA[J]. Current Biotechnology, 2024, 14(4): 594-600.

图/表 8

参考文献 37

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组别	测定浓度/(ng·μL^-1)	实际浓度/(mg·mL^-1)	纯度
TN5-1	297.8	2.97	90%
TN5-2	291.5	2.91	83%
TN5-3	302.3	3.02	93%

组别	测定浓度/(ng·μL^-1)	实际浓度/(mg·mL^-1)	纯度
TN5-1	297.8	2.97	90%
TN5-2	291.5	2.91	83%
TN5-3	302.3	3.02	93%

样本类型	C_t值：原液	C_t值：原液稀释100倍
阳性对照	30.05	32.20
阴性对照	UN	UN
TN5-1	UN	UN
TN5-2	UN	UN
TN5-3	UN	UN

样本类型	C_t值：原液	C_t值：原液稀释100倍
阳性对照	30.05	32.20
阴性对照	UN	UN
TN5-1	UN	UN
TN5-2	UN	UN
TN5-3	UN	UN

样品数量	簇密度	簇通过率	Q30	测序成功率
1 247	1 210	89.5%	89.0%	98.7%
1 374	1 068	91.4%	88.9%	95.7%
1 461	1 264	83.7%	86.6%	97.0%
1 467	1 421	86.7%	87.1%	98.0%
1 473	1 144	89.3%	88.0%	98.1%
1 672	1 366	85.9%	87.2%	97.0%
1 769	1 308	88.8%	88.0%	98.7%
1 789	1 291	85.8%	87.8%	97.5%
1 832	1 358	88.0%	86.5%	97.6%
2 181	1 228	89.4%	87.2%	97.1%
2 198	1 346	86.3%	86.5%	97.3%
2 202	1 105	92.4%	89.5%	98.5%
2 285	1 206	90.3%	89.8%	98.7%
2 418	1 487	85.3%	87.1%	95.1%
2 507	1 225	87.9%	89.8%	94.5%
2 532	1 356	87.6%	91.4%	94.5%
2 643	1 100	89.3%	89.2%	93.1%
2 801	1 212	90.6%	87.7%	93.6%
2 864	1 248	89.7%	89.8%	93.5%
2 980	1 252	89.4%	88.0%	92.1%
2 996	1 162	90.4%	90.3%	92.7%
3 108	1 109	92.4%	90.3%	91.3%
3 809	1 434	85.5%	86.7%	88.2%
4 613	1 330	88.0%	88.4%	78.2%
4 789	1 761	89.0%	91.3%	78.7%

质粒DNA Fast NGS测序方法的开发和应用

Development and Application of Fast NGS Sequencing Method for Plasmid DNA

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对比指标	Sanger测序	Fast NGS
测序反应和纯化耗时	3 h	3 h
测序速度	96个/1.5 h	2 500个/12 h
结果分析耗时	1 h	1 h

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