Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations
Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations
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DOI：
10.1016/j.plabm.2020.e00153
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发表时间：
2020-03-01
影响因子：
1.9
通讯作者：
Lockwood, Christina M.
中科院分区：
文献类型：
--
作者：
Kuo, Ayako J.;Paulson, Vera A.;Lockwood, Christina M.
目的：快速发现具有临床意义的遗传变异已经转化为下一代测序测定，当它们被设计、验证和实施时已经过时。UW-Oncocombinant通过采用能够重新设计的模块化面板来解决这一问题，因为确定了重大改变。我们描述了Oncoclavin版本6（OPXv 6）用于检测340个基因组中的单核苷酸变异（SNV）、插入和缺失（indels）、拷贝数变异（CNV）、结构变异（SV）、微卫星不稳定性（MSI）和肿瘤突变负荷（TMB）的验证。设计：112例不同诊断的样本包括福尔马林固定石蜡包埋组织、新鲜冷冻组织、血浆、外周血、骨髓、唾液和细胞系DNA。从基因组和无细胞DNA制备文库，与定制的xGen Lockdown探针组杂交，并在Illumina平台上测序。结果：SNVs >= 5%等位基因频率的准确率为99%，插入缺失的准确率为98%，SV的准确率为97%，CNVs的准确率为99%，MSI的准确率为100%，TMB的准确率为100%（与以前的Oncoconut版本相比）。文库制备周转时间减少了40%，测序质量提高了2.5倍的平均测序覆盖率和4倍的增加百分比on-target.Conclusions：OPXv 6证明了比以前的UW-Oncoconut版本，包括降低捕获成本，提高测序质量，并减少时间的结果。模块化捕获探针设计还提供了灵活的实验室响应，以解决满足不断发展的分子肿瘤学领域的需求所需的扩展。
Objectives: The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alterations are identified. We describe the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes.Design: One hundred twelve samples with diverse diagnoses were comprised of formalin-fixed-paraffin-embedded tissue, fresh-frozen tissue, plasma, peripheral blood, bone marrow, saliva, and cell-line DNA. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms. Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results.Results: Accuracy was 99% for SNVs >= 5% allele frequency, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB (compared to previous OncoPlex versions). Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target.Conclusions: OPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to results. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology.