Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants. We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH. This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

中文翻译：

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比较匹配的FF和FFPE WGS样本的体细胞变异的比较分析。

在临床实践中尚未常规收集研究级的新鲜冷冻（FF）DNA材料。但是，许多医院都收集并存储福尔马林固定石蜡包埋（FFPE）肿瘤样本。因此，通过增加FFPE样本，可以大大增加全基因组癌症研究的样本量，尽管人工制品的存在可能会混淆变体的调用。为了评估FFPE材料是否可用于队列研究，我们对从相同肿瘤中提取的匹配FF和FFPE全基因组序列（WGS）样品的体细胞SNV进行了深入比较。四个变体调用者（即Strelka2，Mutect2，VarScan2和Shimmer）被用来在来自转移性前列腺肿瘤的匹配FF和FFPE WGS样品上调用体细胞变体。使用这些调用者标识的变体，我们开发了一种启发式方法，可以在灵敏度和精度方面最大化FF与FFPE对应物之间的重叠。然后，在9个匹配的主要样本上验证了提出的变异调用方法。最后，通过比较克隆体和亚克隆体细胞变异的重叠，我们评估了差异的多少可归因于肿瘤内异质性（ITH）。我们首先比较了来自转移性前列腺肿瘤的FF和FFPE样品之间的变异，结果表明FFPE样品中平均回收了FF中50％的检出物，呼叫者之间存在显着差异。使用简单的启发式方法将不同调用方的变体组合在一起，可以提高变体调用的准确性和敏感性。在另外九个匹配的FF-FFPE样本上验证启发式算法，导致F1的平均得分为0.58，并且任何单个呼叫者的表现都超过了。此外，我们可以证明FF和FFPE样本之间的部分差异可以归因于ITH。这项研究表明，使用正确的变异检出策略，可以在FFPE样品中以高精度和高灵敏度回收大多数克隆SNV。这些结果表明，从FFPE材料的WGS衍生的体细胞变异体可用于队列研究。FFPE样品中的大多数克隆SNV可以高精度和高灵敏度回收。这些结果表明，从FFPE材料的WGS衍生的体细胞变异体可用于队列研究。FFPE样品中的大多数克隆SNV可以高精度和高灵敏度回收。这些结果表明，从FFPE材料的WGS衍生的体细胞变异体可用于队列研究。