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中华临床实验室管理电子杂志

中华临床实验室管理电子杂志 ›› 2020, Vol. 08 ›› Issue (02) : 87 -93. doi: 10.3877/cma.j.issn.2095-5820.2020.02.005

所属专题： 文献；

实验研究

人全外显子组测序IDT捕获探针的性能验证
孙明明1, 刘菲菲1, 欧小华2, 胡昌明2, 毛琳琳1, 赵薇薇1,†()   
  1. 1. 510005　广州，广州金域医学检验中心有限公司，临床基因组检测中心；510005　广州，广州金域医学检验集团股份有限公司
    2. 510005　广州，广州金域医学检验中心有限公司，临床基因组检测中心
  • 收稿日期:2019-09-22 出版日期:2020-05-28
  • 通信作者: 赵薇薇
  • 基金资助:
    广州市产业领军人才集聚工程(CXLJTD-201603); 广州市科技计划项目(201604046001)

Performance verification of IDT capture probes for human Whole-exome sequencing

Mingming Sun1, Feifei Liu1, Xiaohua Ou2, Changming Hu2, Linlin Mao1, Weiwei Zhao1,†()   

  1. 1. Clinical Genome Center, KingMed Center for Clinical Laboratory Co., Ltd., Guangzhou 510005, China; Guangzhou KingMed Diagnostics Group Co., Ltd., Guangzhou 510005, China
    2. Clinical Genome Center, KingMed Center for Clinical Laboratory Co., Ltd., Guangzhou 510005, China
  • Received:2019-09-22 Published:2020-05-28
  • Corresponding author: Weiwei Zhao
  • About author:
    Corresponding author: Zhao Weiwei, Email:
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引用本文：

孙明明, 刘菲菲, 欧小华, 胡昌明, 毛琳琳, 赵薇薇. 人全外显子组测序IDT捕获探针的性能验证[J]. 中华临床实验室管理电子杂志, 2020, 08(02): 87-93.

Mingming Sun, Feifei Liu, Xiaohua Ou, Changming Hu, Linlin Mao, Weiwei Zhao. Performance verification of IDT capture probes for human Whole-exome sequencing[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2020, 08(02): 87-93.

目的

探讨已建立的二代测序技术进行人全外显子组测序项目对变异的检出性能。

方法

对4例实验室能力比对验证样本和4例已知结果样本进行全外显子组检测，分析点变异和插入缺失变异，从检测准确度、检测灵敏度、检测特异性、精密度和可报告范围等方面进行性能评估。数据质量控制以目标区域覆盖率、捕获特异性和平均深度为基础。

结果

数据输出量大于8G，实现了目标序列区域99.7%以上的覆盖率，捕获特异性86%以上，平均测序深度100×以上，Q30大于90%，对SNV的检出率达100%，50 bp以下Indel检出率达100%。

结论

本标准操作在验证范围内对变异的检出能力达到应用的要求。

关键词: 全外显子组, 验证, 灵敏度, 特异性, 准确度
Objective

To investigate the detection performance of Whole-exome sequencing (WES) for calling variants by the established second-generation sequencing technology.

Methods

Four CAP proficiency testing samples and 4 known results samples were detected by WES. Point mutations and insertion deletion mutations were analyzed. Detection performance was evaluated in terms of accuracy, sensitivity, specificity, precision and reportable range of calling variants. Data quality control was performed based on coverage of target region, capture specificity and mean depth.

Results

Output data should not be less than 8G. In this case, coverage of target region is over 99.7%. Capture specificity is over 86%. Mean depth is over 100×. Q30 is over 90%, The detection of single nucleotide variations and small insertion and deletion under 50bp is up to 100%.

Conclusion

The standard operating procedure within the scope of validation can meet the application requirement of mutation detection.

Key words: Whole-exome sequencing, Verification, Sensitivity, Specificity, Accuracy
图1 生物信息学分析
表1 原始数据分析质控参数及参考值
Raw Data QC 参考范围 本研究样本数据平均值
Total reads/sample (M) ≥50 67.18
Total base/sample (G) ≥8 9.94
Mapped rate (%) ≥99 99.86
Mean depth of target region (×) ≥100 140.45
Duplication Rate (%) <5 2.91
Capture specificity (%) ≥80 83.99
Coverage of target region (%) ≥99 99.83
Percent of target region ≥20×(%) ≥95 99.63
表2 5例样本8个SNV的NGS检测结果
样本号 样本已知的SNV阳性位点 NGS检测结果
覆盖度(×) 频率(%)
MGL4-14 SMPD1:NM_000543:c.1493G>T(p.R498L) 85 40
GBA:NM_000157:c.1226A>G(p.N409S) 86 54.1
MGL4-18 MCOLN1:NM_020533:c.406-2A>G 22 100
NP15D315 C7:NM_000587:c.281-1G>T 41 24.4
NP15D2229 CDAN1:NM_138477:c.3331C>T(p.R1111*) 116 67
FANCA:NM_000135:c.1108G>T(p.E370*) 113 50.4
NP15D2572 IFIH1:NM_022168:c.2647G>C(p.E883Q) 29 48.3
COL6A3:NM_004369:c.6857A>C(p.N2286T) 45 36.4
表3 6例样本9个Indel的NGS检测结果
样本号 PT/已知结果 NGS检测结果
覆盖度(×) 频率(%)
MGL4-10 HEXA:NM_000520:c.1274-1277dupTATC 61 100
MGL4-13 BLM:NM_000057:c.2207-2212delinsTAGATTC 9 100
MGL4-14 SMPD1:NM_000543:c.1829-1831delGCC 60 36.8
R5806 TSC2:NM_000548:c.4135_4143dupTCGCAGCCC 87 43.4
GAA:NM_000152:c.2024_2026delACA 52 29.4
GAA:NM_000152:c.2312delC 133 37
NP15D315 CD59:NM_203329:c.29delT 47 44.4
CD59:NM_203329:c.283_354del72 85 未检测到
NP15D2572 SLC25A13:NM_014251:c.852_855delTATG 27 29.7
表4 4例CAP PT标本阴性位点的NGS检测结果
样本号 PT结果 NGS检测结果 覆盖度(×) 频率(%)
MGL4-10 ASPA: No variant NM_000049:c.693C>T(p.Y231Y) 56 50
IKBKAP: No variant NM_003640:c.3473C>T(p.P1158L) 36 100
NM_003640:c.3214T>A(p.C1072S) 83 100
NM_003640:c.441G>A(p.Q147Q) 55 100
MGL4-13 GBA: No variant No variant detected - -
SMPD1: No variant
: No variant		 NM_000543:c.138_143delGCTGGC 119 100
NM_000543:c.636T>C(p.D212D) 36 55.6
MGL4-14 BLM: No variant No variant detected - -
MGL4-18 FANCC: No variant
: No variant
: No variant		 N-UTR3 64 100
N-UTR3 81 61.2
N-UTR3 87 58.6
G6PC: No variant No variant detected - -
表5 2例CAP PT标本NGS检测结果说明
样本号 基因 NGS检测结果 千人基因组数据库频率(%) 金域遗传病数据库频率(%) Clinvar(20170130)
MGL4-10 ASPA NM_000049:c.693C>T(p.Y231Y) 20 50 likely benign
IKBKAP NM_003640:c.3473C>T(p.P1158L) 25 51 likely benign
NM_003640:c.3214T>A(p.C1072S) 25 51 likely benign
NM_003640:c.441G>A(p.Q147Q) 8 17.7 likely benign
MGL4-13 SMPD1 NM_000543:c.138_143delGCTGGC 0 29 benign
NM_000543:c.636T>C(p.D212D) 15 0.9 likely benign
表6 4例样本7个SNV两次检测的结果比较
实验号 PT/已知结果 第一次检测 第二次检测
覆盖度(×) 频率(%) 覆盖度(×) 频率(%)
MGL4-14 SMPD1:NM_000543:c.1493G>T(p.R498L) 85 40 92 32.2
GBA:NM_000157:c.1226A>G(p.N409S) 86 54.1 71 59.2
MGL4-18 MCOLN1:NM_020533:c.406-2A>G 22 100 17 94.1
NP15D2229 CDAN1:NM_138477:c.3331C>T(p.R1111*) 116 67 126 58.7
FANCA:NM_000135:c.1108G>T(p.E370*) 113 50.4 98 43.9
NP15D2572 IFIH1:NM_022168:c.2647G>C(p.E883Q) 29 48.3 35 68.6
COL6A3:NM_004369:c.6857A>C(p.N2286T) 45 36.4 71 47.9
表7 3例样本5个Indel两次检测的结果比较
实验号 PT/已知结果 第一次检测 第二次检测
覆盖度(×) 频率(%) 覆盖度(×) 频率(%)
MGL4-10 HEXA:NM_000520:c.1274-1277dupTATC 61 100 39 100
R5806 TSC2:NM_000548:c.4135_4143dupTCGCAGCCC 87 43.4 73 42.5
GAA:NM_000152:c.2024_2026delACA 52 29.4 64 56.2
GAA:NM_000152:c.2312delC 133 37 116 43.4
NP15D2572 SLC25A13:NM_014251:c.852_855delTATG 27 29.7 48 34.8
表8 4例样本7个SNV的两次检测结果比较
实验号 突变内容 第一次检测 第二次检测
覆盖度(×) 频率(%) 覆盖度(×) 频率(%)
MGL4-14 SMPD1:NM_000543:c.1493G>T(p.R498L) 92 32.2 130 41.9
GBA:NM_000157:c.1226A>G(p.N409S) 71 59.2 113 41.1
MGL4-18 MCOLN1:NM_020533:c.406-2A>G 17 94.1 75 100
NP15D2229 CDAN1:NM_138477:c.3331C>T(p.R1111*) 126 58.7 125 42.4
FANCA:NM_000135:c.1108G>T(p.E370*) 98 43.9 122 57.4
NP15D2572 IFIH1:NM_022168:c.2647G>C(p.E883Q) 35 68.6 64 48.4
COL6A3:NM_004369:c.6857A>C(p.N2286T) 71 47.9 98 39.8
表9 3例样本5个Indel的两次检测结果比较
实验号 突变内容 第一次检测 第二次检测
覆盖度(×) 频率(%) 覆盖度(×) 频率(%)
MGL4-10 HEXA:NM_000520:c.1274-1277dupTATC 39 100 73 100
R5806 TSC2:NM_000548:c.4135_4143dupTCGCAGCCC 116 43.4 200 24
GAA:NM_000152:c.2024_2026delACA 73 42.5 109 46.2
GAA:NM_000152:c.2312delC 64 56.2 94 43.6
NP15D2572 SLC25A13:NM_014251:c.852_855delTATG 48 34.8 61 40
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