Department Obstetrics and Gynecology, The Second Affiliated Hospital, Anhui Medical University, Hefei, China.
BGI Genomics, BGI-Shenzhen, Shenzhen, China.
Medicine (Baltimore). 2022 Mar 11;101(10):e28972. doi: 10.1097/MD.0000000000028972.
To evaluate the performance of polymerase chain reaction (PCR)-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.Five NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing deoxyribonucleic acid (DNA) input and library preparation protocol (PCR-based vs PCR-free protocols for library preparation). The depth of coverage and genotype quality of each sample were compared. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes, and copy number variants. We also developed a systematic WGS pipeline (PCR-free) for the analysis of 11 clinical cases.In general, NA12878-2 (PCR-free WGS) showed better depth of coverage and genotype quality distribution than NA12878-1 (PCR-based WGS). With a mean depth of ∼40×, the sensitivity of homozygous and heterozygous single nucleotide polymorphisms (SNPs) of NA12878-2 showed higher sensitivity (>99.77% and >99.82%) than NA12878-1, and positive predictive value exceeded 99.98% and 99.07%. The sensitivity and positive predictive value of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and copy number variants are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. All the 19 previously confirmed variants in 11 clinical cases were successfully detected by our WGS pipeline (PCR free).Different experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.
为了评估聚合酶链反应（PCR）-免费全基因组测序（WGS）在临床诊断中的性能，从而揭示实验参数如何影响变异检测。使用 MGISEQ-2000 对 5 个 NA12878 样本进行测序。NA12878 样本进行了 WGS 测序，脱氧核糖核酸（DNA）输入和文库制备方案不同（文库制备的 PCR 与 PCR 自由方案）。比较了每个样本的覆盖深度和基因型质量。测量了每个样本的性能，包括灵敏度、疾病相关基因的覆盖深度和广度以及拷贝数变异。我们还为 11 个临床病例开发了一个系统的 WGS 分析流程（PCR 免费）。
一般来说，NA12878-2（PCR 免费 WGS）的覆盖深度和基因型质量分布优于 NA12878-1（PCR 基于 WGS）。NA12878-2 的平均深度约为 40×，其纯合和杂合单核苷酸多态性（SNP）的灵敏度（>99.77%和>99.82%）高于 NA12878-1，阳性预测值超过 99.98%和 99.07%。NA12878-2（PCR 免费 WGS）中纯合和杂合插入缺失的灵敏度和阳性预测值均有较大提高，而 NA12878-1 则没有。与具有基于 PCR 的文库制备方案的样品相比，具有 PCR 免费文库制备方案的样品的疾病相关基因和拷贝数变异的覆盖范围略宽。DNA 输入也会影响 PCR 免费 WGS 样品中变异检测的性能。通过我们的 WGS 分析流程（PCR 免费）成功检测到 11 个临床病例中 19 个先前证实的变异。
不同的实验参数可能会影响临床 WGS 的变异检测。临床科学家应该了解不同 WGS 方法的变异检测灵敏度范围，这在解释和提供临床报告时将非常有用。
