PO.MCB08.03 · 分子与细胞生物学

来自低起始量FFPE样本的高保真全基因组测序：通过卓越的变异检测和均一覆盖实现精准的肿瘤指导型MRD检测设计

High-fidelity whole genome sequencing from low-input FFPE samples: Enabling accurate tumor-informed MRD assay design through superior variant detection and uniform coverage

编号 3241 展板 6 时间 4/20 02:00–05:00 区域 Section 22 主讲 Eugenio Daviso

分会场 Genomic Profiling to Understand Cancer Biology

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作者与单位 Authors & Affiliations

Vanessa Process, Sushant Khanal, Madan Ambavaram, Sameer Vasantgadkar, Luca Beker, Alaina Villarreal, Jose Gil, Andrew Laneville, Martina Werner, Greg Endress, Ulrich Thomann, Eugenio Daviso

Covaris, LLC, Woburn, MA

摘要 Abstract

中文摘要

引言：肿瘤指导型微小残留病（MRD）监测依赖于从原发肿瘤组织中精确识别患者特异性体细胞变异。然而，这些样本常以福尔马林固定石蜡包埋（FFPE）蜡块的形式存档，其中DNA损伤、片段化和化学修饰（如胞嘧啶脱氨基）会损害测序质量。不准确的基线分析可能导致将假象选为追踪靶标，或遗漏关键的亚克隆突变，最终降低MRD检测的灵敏度。在此，我们展示了一种针对低起始量FFPE样本优化的高保真全基因组测序（WGS）工作流程，该流程可最大限度地减少扩增偏倚和假象，确保生成稳健的MRD靶标选择所需的可靠基因组图谱。方法：使用Covaris truXTRAC® FFPE SMART试剂盒结合自适应聚焦声波（AFA®）技术从FFPE处理的NA12878细胞（FF12878）中提取基因组DNA，以确保有效去除石蜡并进行再水化。使用truCOVER® WGS文库制备试剂盒（含扩增）构建WGS文库。该工作流程使用从1 ng到50 ng的DNA起始量进行评估。文库性能以高质量基因组DNA（NA12878）起始量（0.1 ng - 50 ng）为基准进行比较。文库在Illumina NovaSeq™ X Plus上测序。使用GIAB真值集对数据进行分析，评估覆盖均一性、重复率以及SNP和INDEL的变异检出准确性（F1分数）。结果：该优化的工作流程展现出卓越的灵敏度，可从少至1 ng的FFPE DNA生成高复杂度文库。对MRD设计至关重要的是，该方法表现出最小化的GC偏倚，无论起始量多少，在504个临床相关癌症基因（TSO500 panel）上均保持归一化覆盖度集中于1.0左右。这种均一性确保了难以测序区域中潜在的追踪突变不会被遗漏。变异检出分析显示，该工作流程有效克服了FFPE诱导的损伤；FFPE样本（5 ng）中SNP和INDEL的F1分数与高质量gDNA相当（INDEL >0.91，SNP >0.97）。此外，重复率得到了显著控制（5 ng FFPE约为19%），最大化了验证纵向追踪所必需的低频主干突变所需的独特读段深度。结论：我们验证了一种稳健的WGS工作流程，可释放存档FFPE组织用于高保真基因组分析，克服了低起始量和DNA损伤这两大传统障碍。通过提供卓越的覆盖均一性和高变异检出准确性，该解决方案提供了设计高度特异性的肿瘤指导型MRD检测所需的精确基因组基线。

查看英文原文 English abstract

Introduction: Tumor-informed Minimal Residual Disease (MRD) monitoring relies on the precise identification of patient-specific somatic variants from primary tumor tissue. However, these samples are frequently archived as Formalin-Fixed Paraffin-Embedded (FFPE) blocks, where DNA damage, fragmentation, and chemical modifications (e.g., cytosine deamination) compromise sequencing quality. Inaccurate baseline profiling can lead to the selection of artifacts as tracking targets or the omission of critical sub-clonal mutations, ultimately reducing MRD assay sensitivity. Here, we present a high-fidelity Whole Genome Sequencing (WGS) workflow optimized for low-input FFPE samples that minimizes amplification bias and artifacts, ensuring the generation of reliable genomic maps necessary for robust MRD target selection. Methods: Genomic DNA was extracted from FFPE-processed NA12878 cells (FF12878) using the Covaris truXTRAC® FFPE SMART Kit with Adaptive Focused Acoustics (AFA®) technology to ensure active paraffin removal and rehydration. WGS libraries were constructed using the truCOVER® WGS Library Prep Kit with Amplification. The workflow was evaluated using DNA inputs ranging from 1 ng to 50 ng. Library performance was benchmarked against high-quality genomic DNA (NA12878) inputs (0.1 ng - 50 ng). Libraries were sequenced on an Illumina NovaSeq™ X Plus. Data was analyzed for coverage uniformity, duplication rates, and variant calling accuracy (F1 scores) for SNPs and INDELs using the GIAB truth set. Results: The optimized workflow demonstrated exceptional sensitivity, generating high-complexity libraries from as little as 1 ng of FFPE DNA. Critical for MRD design, the method exhibited minimized GC bias, maintaining normalized coverage centered around 1.0 across 504 clinically relevant cancer genes (TSO500 panel), irrespective of input amount. This uniformity ensures that potential tracking mutations in difficult-to-sequence regions are not missed. Variant calling analysis revealed that the workflow effectively overcomes FFPE-induced damage; F1 scores for SNPs and INDELs in FFPE samples (5 ng) were comparable to those of high-quality gDNA (>0.91 for INDELs and >0.97 for SNPs). Furthermore, duplication rates were significantly controlled (~19% for 5 ng FFPE), maximizing the unique read depth required to validate low-frequency truncal mutations essential for longitudinal tracking. Conclusions: We have validated a robust WGS workflow that unlocks archival FFPE tissues for high-fidelity genomic profiling, overcoming traditional barriers of low input and DNA damage. By delivering superior coverage uniformity and high variant calling accuracy, this solution provides the precise genomic baseline required for designing highly specific tumor-informed MRD assays

利益披露 Disclosure

V. Process, None.. S. Khanal, None.. M. Ambavaram, None.. S. Vasantgadkar, None.. L. Beker, None.. A. Villarreal, None.. J. Gil, None.. A. Laneville, None.. M. Werner, None.. G. Endress, None.. U. Thomann, None.. E. Daviso, None.

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