Quality control and proficiency testing in next-generation sequencing of inherited genetic mutations

doi:10.3969/j.issn.1673-8640.2025.02.003

Abstract

Abstract:

Next-generation sequencing（NGS） provides technical support for the precise detection of genetic mutations associated with hereditary diseases and their diagnosis. However，the accuracy and validity of NGS are susceptible to various factors，including sample quality，experimental procedures，instrument performance and datum analysis processes. Therefore，clinical laboratories must implement comprehensive quality control measures to guarantee the validity of the detection results. This review focuses on the quality control strategies and proficiency testing（PT） methods in the NGS workflow，providing a reference for standardized laboratory management of NGS.

Key words: Next-generation sequencing, Quality control, Proficiency testing

CLC Number:

R446.1

WANG Yange, YANG Jiyun, ZHOU Yu, JIANG Li. Quality control and proficiency testing in next-generation sequencing of inherited genetic mutations[J]. Laboratory Medicine, 2025, 40(2): 114-120.

Figures/Tables 3

References 33

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方法	特点	局限性
TGS	1）通过对特定目标基因区域的高效富集和深度测序实现精准检测；2）敏感性和特异性高	1）仅能检测预设的目标基因，无法发现新的致病基因或未知变异；2）基因Panel需不断更新，以纳入新的相关基因
WES	1）靶向编码蛋白的外显子区域，同时覆盖部分基因调控区；2）检测已知基因变异，并发现新的候选致病基因；3）常用于单基因遗传病的诊断	1）仅覆盖约2%的基因组区域，无法检测非编码调控区的关键序列；2）临床诊断率有限
WGS	1）全面覆盖基因组，包括非编码区；2）可检测多种变异（单核苷酸变异、插入、缺失、基因组结构变异等）；3）不受捕获和扩增限制	1）覆盖深度低于WES；2）成本较高，临床应用受限

方法	特点	局限性
TGS	1）通过对特定目标基因区域的高效富集和深度测序实现精准检测；2）敏感性和特异性高	1）仅能检测预设的目标基因，无法发现新的致病基因或未知变异；2）基因Panel需不断更新，以纳入新的相关基因
WES	1）靶向编码蛋白的外显子区域，同时覆盖部分基因调控区；2）检测已知基因变异，并发现新的候选致病基因；3）常用于单基因遗传病的诊断	1）仅覆盖约2%的基因组区域，无法检测非编码调控区的关键序列；2）临床诊断率有限
WGS	1）全面覆盖基因组，包括非编码区；2）可检测多种变异（单核苷酸变异、插入、缺失、基因组结构变异等）；3）不受捕获和扩增限制	1）覆盖深度低于WES；2）成本较高，临床应用受限

PT方式	描述	优缺点	挑战
MBPT	评估实验室NGS操作的全流程，包括样本处理、文库制备、测序和生物信息学分析，确保检测的可靠性和准确性	优点：全面评估检测流程，能够模拟不同VAF（变异等位基因频率），并进行真实评估缺点：受DNA样本中的变异种类和VAF模拟的灵活性限制，评估范围有限	需要复杂的技术（如质粒、基因编辑技术）扩展应用范围
ISPT	通过计算机模拟生成变异样本序列文件，专注评估NGS中的生物信息学数据分析过程，包括变异检测、注释	优点：灵活性高，适用于跨实验室、跨平台的评估，能够模拟复杂变异，并评估不同VAF 缺点：仅限于生物信息学分析，无法覆盖NGS全流程	可能存在偏差和伪影，依赖专家管理和处理模拟序列文件

PT方式	描述	优缺点	挑战
MBPT	评估实验室NGS操作的全流程，包括样本处理、文库制备、测序和生物信息学分析，确保检测的可靠性和准确性	优点：全面评估检测流程，能够模拟不同VAF（变异等位基因频率），并进行真实评估缺点：受DNA样本中的变异种类和VAF模拟的灵活性限制，评估范围有限	需要复杂的技术（如质粒、基因编辑技术）扩展应用范围
ISPT	通过计算机模拟生成变异样本序列文件，专注评估NGS中的生物信息学数据分析过程，包括变异检测、注释	优点：灵活性高，适用于跨实验室、跨平台的评估，能够模拟复杂变异，并评估不同VAF 缺点：仅限于生物信息学分析，无法覆盖NGS全流程	可能存在偏差和伪影，依赖专家管理和处理模拟序列文件