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

doi:10.3877/cma.j.issn.2095-5820.2020.02.005

Abstract

Abstract:

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

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.

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Figures/Tables 10

References 17

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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

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

样本号	样本已知的SNV阳性位点	NGS检测结果
样本号	样本已知的SNV阳性位点	覆盖度(×)	频率(%)
MGL4-14	SMPD1:NM_000543:c.1493G>T(p.R498L)	85	40
MGL4-14	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
NP15D2229	FANCA:NM_000135:c.1108G>T(p.E370*)	113	50.4
NP15D2572	IFIH1:NM_022168:c.2647G>C(p.E883Q)	29	48.3
NP15D2572	COL6A3:NM_004369:c.6857A>C(p.N2286T)	45	36.4

样本号	样本已知的SNV阳性位点	NGS检测结果
样本号	样本已知的SNV阳性位点	覆盖度(×)	频率(%)
MGL4-14	SMPD1:NM_000543:c.1493G>T(p.R498L)	85	40
MGL4-14	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
NP15D2229	FANCA:NM_000135:c.1108G>T(p.E370*)	113	50.4
NP15D2572	IFIH1:NM_022168:c.2647G>C(p.E883Q)	29	48.3
NP15D2572	COL6A3:NM_004369:c.6857A>C(p.N2286T)	45	36.4

样本号	PT/已知结果	NGS检测结果
样本号	PT/已知结果	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
NP15D315	CD59:NM_203329:c.283_354del72	85	未检测到
NP15D2572	SLC25A13:NM_014251:c.852_855delTATG	27	29.7

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