高通量测序文库质量控制技术研究进展

doi:10.16288/j.yczz.23-262

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Hereditas(Beijing) ›› 2024, Vol. 46 ›› Issue (2): 140-148.doi: 10.16288/j.yczz.23-262

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Progress on the quality control technology of next generation sequencing library

Mengnan Cui(), Yan Guo, Yarong Wu, Guangqian Pei, Yujun Cui()

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China

Received:2023-10-23 Revised:2023-12-22 Online:2024-02-20 Published:2024-01-05
Contact: Yujun Cui E-mail:mengncui@163.com;cuiyujun.new@gmail.com

Abstract

Abstract:

As a key supporting technology in the fields of life sciences and medicine, high-throughput sequencing has developed rapidly and become increasingly mature. The workflow of this technology can be divided into nucleic acid extraction, library construction, sequencing, and data analysis. Among these, library construction is a pivotal step that bridges the previous and subsequent stages. The effectiveness of library construction is contingent on the quality of upstream samples and also impacts the data analysis following sequence data output. The selection and implementation of library construction quality control techniques are crucial for enhancing the reliability of results and reducing errors in sequencing data. This review provides an in-depth discussion of library construction quality control techniques, summarizing and evaluating their principles, advantages and disadvantages, and applicability. It also discusses the selection of relevant technologies in practical application scenarios. The aim is to offer theoretical foundations and references for researchers, disease prevention and control personnel, and others when choosing library quality control techniques, thereby promoting the quality and efficiency of high-throughput sequencing work.

Key words: next generation sequencing library, library concentration, size distribution of the library, quality control, technology analysis

Mengnan Cui, Yan Guo, Yarong Wu, Guangqian Pei, Yujun Cui. Progress on the quality control technology of next generation sequencing library[J]. Hereditas(Beijing), 2024, 46(2): 140-148.

Figures/Tables 3

Fig. 1

Table 1

Table 2

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类别	紫外吸收技术	荧光染料技术	qPCR(SYBR-Green)	qPCR(TaqMan)	ddPCR
典型仪器	NanoDrop	Qubit	ABI7500	ABI7500	qx200
投入量(μL)	1	1~20	2	2	2
定量范围	2~3700 ng/μL^a	10 pg/μL~100 ng/μL^b	0.00083~8.3pmol/L^[18]	0.068~6.8 pmol/L^[18]	1~10⁵个拷贝^c
单次检测量	1	1	≤96	≤96	≤96
单次检测时间	30 s	1 min	2.5 h	2.5 h	微滴生成<2 min/ 8 个，PCR反应时间2 h，微滴检测< 10 min/个
试剂成本/单样品 (美元)	<0.5	0.7	3.0	5.9	7~9.8
优点	操作简单、检测速度快、成本低	操作简单、成本较低，可以特异性区分DNA、RNA，检测灵敏度高、重复性好	不需要设计荧光探针，灵活，成本较低，能区分文库两端是否连接接头	需要设计荧光探针，成本较低，能区分文库两端是否连接接头	绝对定量，不依赖于具有特定片段大小的标准品，不依赖于校准物的扩增效率，准确度更高、置信度和可重复性更好
缺点	对DNA、RNA、蛋白质没有选择性，受杂质影响大，精度差	无法区分文库两端是否连接接头	不能区分接头二聚体，依赖于生物分析仪对文库片段大小的检测	不能区分接头二聚体，依赖于生物分析仪对文库片段大小的检测	成本较高

类别	琼脂糖凝胶电泳技术	微流控芯片电泳技术
典型仪器		Agilent 2100 Bioanalyzer
投入量		1 μL
分离片段范围	与琼脂糖凝胶配制浓度相关	25 bp~12 kb
灵敏度	与作为DNA分子量标准有关	0.1 ng DNA
单次检测量	与制胶时的孔容量有关	12
单次检测时间		3 min
优点	原理、操作方法等较简单，成本较低	所需进样量、试剂量等少、人工操作时间短、操作安全、数据可视化、分析自动化、准确度高、重复性好、灵敏度高
缺点	分离精度较低、操作繁琐、自动化程度低	试剂、芯片价格较昂贵

Progress on the quality control technology of next generation sequencing library

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