Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.
Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.
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DOI：
10.1371/journal.pone.0127353
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Schully SD
中科院分区：
文献类型：
--
作者：
Carrick DM;Mehaffey MG;Sachs MC;Altekruse S;Camalier C;Chuaqui R;Cozen W;Das B;Hernandez BY;Lih CJ;Lynch CF;Makhlouf H;McGregor P;McShane LM;Phillips Rohan J;Walsh WD;Williams PM;Gillanders EM;Mechanic LE;Schully SD
下一代测序（NGS）技术用于检测肿瘤中的体细胞突变和研究生殖系变异。大多数NGS研究使用从全血或新鲜冷冻组织中分离的DNA。然而，福尔马林固定石蜡包埋（FFPE）组织是最广泛使用的临床标本之一。它们作为NGS DNA来源的潜在效用将大大增强基于人群的癌症研究。虽然初步研究表明FFPE组织可用于NGS，但需要确定在基于人群的研究中使用存档FFPE标本的可行性以及储存时间对这些标本的影响。我们进行了一项研究，以确定来自监测、流行病学和最终结果（SEER）登记处残留组织储存库（RTR）的存档FFPE高级别卵巢浆液性腺癌中的DNA是否以足够的数量和质量用于NGS检测。从三个SEER RTR研究中心获得了59个FFPE组织，储存时间为3 - 32年。提取DNA，定量，质量评估，并进行全外显子组测序（WES）。DNA提取后，59份标本中的58份（98%）产生了DNA，并继续进行文库生成步骤，然后进行WES。储存较长时间的样本具有显著较低的目标区域覆盖率（每10年降低6%，95%CI：3-10%）和较低的平均读取深度（每10年降低40倍，95%CI：18-60），尽管获得了足够质量和数量的WES数据用于数据挖掘。总体而言，无论储存时间如何，90%（53/59）的标本提供了可用的NGS数据。该可行性研究表明，在不同储存时间和不同储存条件下从SEER登记系统获得的FFPE标本是NGS的有希望的DNA来源。
Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.