Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.
Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.
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DOI：
10.1371/journal.pone.0144162
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Shin YK
中科院分区：
文献类型：
--
作者：
Oh E;Choi YL;Kwon MJ;Kim RN;Kim YJ;Song JY;Jung KS;Shin YK
福尔马林固定石蜡包埋（FFPE）已经是一个标准的样品制备方法几十年来，档案FFPE样品仍然是非常有用的资源。尽管如此，由于DNA质量差和人工序列改变，使用下一代测序在癌症基因组分析中使用FFPE样品一直具有挑战性，下一代测序是在核苷酸水平上鉴定基因组改变的强大技术。在这项研究中，我们对来自4名癌症患者的组织的匹配冷冻样品和FFPE样品进行了全外显子组测序，并比较了从这些样品中获得的下一代测序数据。从2种类型样本中获得的数据之间的主要差异是FFPE样本中的插入物尺寸较短和人工碱基改变。FFPE样品中高比例的短插入片段导致重叠的配对读段，这可能导致高估某些变体; FFPE样品中>20%的插入片段被双重测序。在FFPE样品的测序数据中发现了大量的软剪切读段，并且约30%的总碱基是软剪切的。仅在FFPE样品中观察到人工碱基改变，C>T和G>A，并且当去除测序错误时，改变率范围为200至1，200/1 M碱基。尽管FFPE样本中的高置信度突变调用与冷冻样本中的突变调用兼容，但应谨慎对待伪影，尤其是低置信度调用。尽管有明显观察到的伪影，档案FFPE样品可以是基于全外显子组测序的癌症研究中发现或验证生物标志物的良好资源。
Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.