PO.MCB08.04 · 分子与细胞生物学
使用LinkPrep从前列腺FFPE活检中准确检测DNA变异和ecDNA
Accurate detection of DNA variants and ecDNA from prostate FFPE biopsies using LinkPrep
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
在福尔马林固定石蜡包埋（FFPE）肿瘤活检中同时检测基因组变异和结构重排仍具挑战性，且缺乏全面的临床应用价值评估。在此，我们对20例前列腺癌样本开展了新颖的Dovetail®-FFPE（LinkPrep），这是一种不受限制性内切酶偏倚影响的体外转座Hi-C检测。这些样本包括5例肿瘤旁正常组织（良性）、10例原发局限性和10例转移性去势抵抗性前列腺癌（mCRPC）患者样本。通过利用患者和位点特异性的自有配对新鲜冷冻（FF）全基因组测序（WGS）、外显子捕获测序、转录组测序，以及一个公开可用的80例mCRPC FF Hi-C样本数据队列，我们对FFPE LinkPrep文库制备和测序性能进行了全面评估。我们的评估涵盖多个方面，包括三维基因组接触检测、单核苷酸变异（SNV）/插入缺失（indel）检出、拷贝数变异分析、结构变异（SV）检测、ecDNA检测和区室（compartment）界定。我们的分析显示，FFPE样本在mCRPC肿瘤中实现了良好的接触捕获效率，略低于但可与FF Hi-C数据相媲美（Wilcoxon检验p=0.33）。在SNV/INDEL检出方面，以FF WGS或外显子捕获测序作为金标准，基于两种不同的变异检出算法，该检测在五个样本中的平均召回率达到97%。通过与2例FF WGS、4例转录组和80例外部Hi-C数据进行比较，我们进一步证明FFPE样本能够准确识别全基因组拷贝数变异和染色质区室。此外，我们开发了一套稳健的SV检出流程，其中精心设计了适用于FFPE样本的去噪步骤，能够准确检测前列腺癌中常见的结构变异，如TMPRSS2-ERG融合（6/7）和FOXA1突变（2/2）。最后，通过应用新开发的Hi-C特异性染色体外DNA（ecDNA）检测流程，我们在一个样本中鉴定出AR ecDNA，这一结果得到了配对FF WGS AmpliconSuite结果的支持。总体而言，这一全面的分析和性能评估表明，FFPE LinkPrep能够准确捕获DNA水平的变异，并在FFPE临床样本中的应用方面具有强大潜力。
查看英文原文 English abstract
Detecting genomic variants and structural rearrangements in formalin-fixed paraffin-embedded (FFPE) tumor biopsies simultaneously remains challenging and lacks comprehensive clinical utility assessments. Here we performed the novel Dovetail®-FFPE (LinkPrep), an in vitro transposition Hi-C assay free from restriction enzyme bias to 20 prostate cancer samples. These included 5 normal adjacent to tumor (benign), 10 primary localized, and 10 metastatic castration-resistant prostate cancer (mCRPC) patient samples. By leveraging patient and site-specific in-house matched fresh-frozen (FF) whole-genome sequencing (WGS), exome capture sequencing, transcriptomics sequencing, and a publicly available cohort of 80 mCRPC FF Hi-C samples data, we performed a comprehensive evaluation on FFPE LinkPrep library preparation and sequencing performance. Our evaluation covered multiple aspects including 3D genome contact detection, single nucleotide variant (SNV)/indel calling, copy number variation analysis, structural variant (SV) detection, ecDNA detection and compartments definition.Our analysis shows that FFPE sample achieves good contact capture efficiency that is slightly lower but comparable performance (Wilcoxon test p=0.33) to the FF Hi-C data for mCRPC tumors. For SNV/INDEL calling, using FF WGS or exome capture sequencing as the gold standard, the assay reached a mean recall rate of 97% across five samples based on two different variant- calling algorithms. By comparing with 2 FF WGS, 4 transcriptomic and 80 external Hi-C data, we further demonstrated that FFPE samples can accurately identifies whole-genome copy number variations and chromatin compartments. Additionally, we developed a robust SV-calling pipeline with carefully designed denoising steps suitable for FFPE samples that accurately detected common structural variants in prostate cancer, such as TMPRSS2-ERG fusions (6/7) and FOXA1mutations (2/2). Lastly, by applying the new developed Hi-C specific pipeline for extrachromosomal DNA (ecDNA) detection, we identified AR ecDNA in one sample, which was supported by matched FF WGS AmpliconSuite result. Overall, the comprehensive analysis and performance evaluation demonstrates that FFPE LinkPrep accurately captures DNA-level variants and holds strong potential for application in FFPE clinical samples.
利益披露 Disclosure
S. Li, None.