NGS在肿瘤临床基因检测中的应用1

临床NGS基因检测的技术策略^[1,3]

Clinical Next-Generation Sequencing (NGS) Test Types

临床中的NGS基因检测可以设计为针对一组选定的基因，外显子组(所有已知的基因，大约2%的基因组)，或整个基因组。但无论选何种策略，对于临床来说终极目的还是预测和寻根问药（专业点说，寻找与临床表型相关的基因变异，进而指导患者使用何种化疗药、靶向药、评估预后、患病风险等），偏离这个方向对临床意义不大。

whole-genome sequencing (WGS)

测大约30亿个碱基对的DNA序列，内含子和基因间区等大量功能未知区域均会被覆盖，获得大量未知变异信息；检测的变异类型多样，SNV、Indels、SV、CNV、基因重排等；更好的测序覆盖度和一致性；缺乏先验信息，很难确定变异是否是致病的，解读非常困难；测序费用高（测100x深度下，费用是WES的3到5倍）；数据分析，存储所需资源更多。

whole-exome sequencing (WES)

测大约5千万个碱基对的DNA序列，包含所有的编码区、5'UTR、及3'UTR区域（ coding exons and exon-intron boundaries），大约占基因组的的2%，大约包含人类已知的20000个基因，至少包含了基因组中致病变异的85%；实际测序时只有90-95%外显子区域被测到（二代测序也是有弊端的，不是所有都能测，见下图）、在高/低GC区域会出现GC-bias和探针捕获效率低，导致该区域出现测序深度低的现象；150X测序下，费用是panel的5到10倍，和WGS一样会测到一些VUS变异（与当前疾病无关的变异，解读如果不恰当会给病人带来心理负担，往往需要增加成本来验证这些无关变异）。

Targeted-panel sequencing（Gene panel）

针对病人某类疾病密切关联的基因设计（这一步需要花费大量精力了解患者表型和可能的相关基因），只需要测几千到几百万个碱基，包含几个到几百个疾病已知相关基因，更经济；测序深度高，成本低；基因少且注释相对完整，解读更容易，检测更高效；相比于WGS和WES，panel不能用来发现新基因。

Single-Gene Testing

测定某种疾病的直接致病基因，如先天性软骨发育不全（achondroplasia, ACH, OMIM: 100800）99%是是由FGFR3基因 c.1138G>A突变所致，Single gene sequencing可以很好的应对这种情况；这类测序通量低；准确率高（常作为WES和panel的验证金标准）。

临床NGS基因检测的突变类型

并非所有的序列都能同样被NGS测序覆盖(表29.2)，因为该技术存在局限性，使得一些突变难以被检测到^[3]。

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基因检测技术策略的比较^[1]

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临床NGS基因检测的实验室流程^[1]

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临床NGS基因检测的生信分析

用于突变检测的分析软件和流程众多，常规的流程如图。以检测肿瘤突变为例，存在很多基于不同检测策略检测不同突变类型的分析流程，如基于WGS、WES和Gene Panel技术检测胚系或(和)体系突变(SNV和indel)的流程^[6-9]，基于WGS检测CNV的流程^[10]，基于WGS检测SV的流程^[11]；但无论哪种分析流程，在用于临床实践之前都需要进行验证^[12]。

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临床NGS基因检测的报告解读^[13-15]

肿瘤NGS基因检测，“报告解读”是关键！它是精准医疗的关键“临门一脚”！对基因检测的需求越来越多；基因检测过程和分析变得越来越复杂；需要对基因检测结果进行个体化临床解读，特别是NGS大panel或WES/WGS；基因检测报告必须写得清楚，报告解读必须通俗易懂，让非遗传学专家，如临床医生或患者也能看懂。

体细胞变异注释及临床解读

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胚系突变分级注释

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参考资料：

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11. Neerman N, Faust G, Meeks N, et al. A clinically validated whole genome pipeline for structural variant detection and analysis. BMC Genomics. 2019;20(Suppl 8):545. Published 2019 Jul 16.

12. Roy S , Coldren C , Karunamurthy A , et al. Standards and Guidelines for Validating Next-Generation Sequencing Bioinformatics Pipelines: A Joint Recommendation of the Association for Molecular Pathology and the College of American Pathologists[J]. Journal of Molecular Diagnostics, 2018:4-27.

13. https://www.seqchina.cn/12736.html

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