参考资料
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets)
A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology
Tumour lineage shapes BRCA-mediated phenotypes
方法介绍
样本准备
选择标准
- 样本肿瘤细胞含量超过20% ；
- 针对MSI检测，肿瘤细胞含量应该超过25% ；
- DNA需要进行质控和富集，进行方法学测试的DNA 需要达到100ng~250ng；
- 平均插入片段（Average fragment）应该在200bp左右；
- 建库前应该存放在-20℃ （The DNA can be stored at 37°C for 10-20 minutes, stored at 2–8°C for 24 hours, or at –20°C for longer periods）。
文库构建
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表现评估
Determination of pipeline thresholds
Requirements on exon coverage were established
不同的测序深度下，观测到的变异频率和理论变异频率之间会存在波动，导致检测结果和理论结果不一致（但理论上大概率会处在某个范围内，理论计算如下表 ：
| 突变实际频率 | 置信区间 | 测序深度 | 检测值范围 |
|---|---|---|---|
| 10% | 95 | 500 | 7.5% ~ 13% |
| 10% | 95 | 100x | 5.0% ~ 17.6% |
为了验证这个理论，使用了10个 正常的FFPE 进行测试，实际测试结果如下：
| 突变实际频率 | 置信区间 | 测序深度 | 检测值范围 |
|---|---|---|---|
| 10% | 95 | 平均深度 480x | 5.0% ~ 13.9% |
测试结果和预测结果相对比较一致。
这个数据支持我们检测10%的目标变异时，使用5%作为检测的阈值 。
Requirements on sample coverage
Based on the calculations, 98% of exons can be expected to be sequenced to coverage greater than 100X, when mean sample coverage is 185X (0.54* 185X = 100X). (A 100X minimum coverage threshold per exon is required based on the power calculations, which showed 100X coverage was necessary to call mutations with true underlying mutation frequency 10% or greater, with 95% power at an alpha level of 0.05).
To be conservative, a threshold of 200X on mean sample coverage is used to determine if a sample is sequenced to sufficient depth for subsequent analysis. A sample is flagged as being at increased risk of false negatives if its mean coverage is below 200X.