Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens
Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens
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DOI：
10.1016/j.jmoldx.2013.05.004
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Duncavage, Eric J.
中科院分区：
文献类型：
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作者：
Spencer, David H.;Sehn, Jennifer K.;Duncavage, Eric J.
下一代测序（NGS）已成为临床实验室检测遗传变异的强大技术。可以使用来自FFPE组织的DNA进行NGS，但尚不清楚此类标本是否真正等同于用于NGS应用的未固定组织。为了解决这个问题，我们使用来自16对新鲜冷冻和常规FFPE肺腺癌标本的DNA对27个癌症相关基因进行了杂交捕获富集和多重Illumina NGS，并对来自每种样本类型的序列数据进行了广泛的比较。该分析揭示了FFPE和冷冻样品之间的微小但可检测的差异。与冷冻样品相比，FFPE样品的NGS数据具有更小的文库插入大小，更大的覆盖率变异性，以及在CpG二核苷酸处最明显的C至T转换的增加，表明DNA甲基化和福尔马林诱导的变化之间的相互作用;然而，错误率，文库复杂性，富集性能和覆盖率统计没有显著差异。配对样本之间碱基识别的比较显示出> 99.99%的一致性，检测到的单核苷酸变异的一致性为96.8%，与来自正交单核苷酸多态性阵列平台的基因型相比，NGS数据的准确性为>98%。本研究表明，FFPE样本的常规处理对NGS数据的影响可检测但可忽略不计，这些样本可以作为临床NGS检测的可靠底物。
Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE Lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.