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Title: [Comparison of different massive parallel sequencing platforms for mutation profiling in formalin-fixed and paraffin-embedded samples].
Author: Jiang RR, Wang YJ, Teng XD, Xiao L, Bu H, Ye F.
Journal: Zhonghua Bing Li Xue Za Zhi; 2018 Aug 08; 47(8):591-596. PubMed ID: 30107663.
Abstract:
Objective: To compare the performance of Miseq and Ion Torrent PGM platforms and library construction method for next-generation sequencing (NGS) technology for formalin-fixed and paraffin-embedded (FFPE) samples. Methods: A total of 204 FFPE cancer samples including 100 non-small cell lung cancers at the First Affiliated Hospital of Zhejiang University, and 104 colorectal cancers at West China Hospital of Sichuan University were retrospectively selected from January 2013 to December 2016. By using the same samples, DNA was extracted, and the same amount of DNA was used for library construction with the same kit, and sequenced on Miseq and Ion Torrent PGM respectively, after passing the quality control. Any discordant mutations between two platforms were validated by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR) method and Sanger sequencing. Results: A total of 204 FFPE samples were included and 197 samples were successfully analyzed by both platforms. The number of reads generated by the samples on Miseq platform sequencing was higher than PGM platform (median 391 634 vs. 298 030, P<0.01). Alignment with human reference genome showed that the mapping rate of Miseq platform was higher than PGM platform (median 100.0% vs. 99.7%, P<0.01). The median sequence depth of samples on Miseq was higher than PGM platform (median 853× vs. 698×, P<0.01). A total of 236 mutations were detected by two platforms, of which 221 were detected on both platforms, with a 93.6% concordance. Miseq platform detected 11 mutations not detected on PGM platform, while PGM platform detected 4 more mutations not detected on Miseq platform. With validation by ARMS-PCR and Sanger sequencing, Miseq platform was more reliable for low-frequency mutations. The main reasons for the discordant mutations between two platforms were that mutation frequency on undetected platform was lower than mutation reporting range (5%) and FFPE samples were stored for a long time. Conclusions: Compared with PGM, Miseq platform shows higher sequencing quality in terms of the number of reads, alignment results and coverage depth, and the test results are more reliable. In clinical practice, the appropriate platform should be chosen based on sample size and actual throughput requirements to aid in the molecular characterization of tumors. 目的： 应用甲醛固定石蜡包埋(FFPE)标本，以相同的建库方法，比较二代测序(NGS)技术Illumina公司的Miseq和Thermo公司的Ion Torrent PGM两个平台的性能和一致性，探讨临床应用价值。 方法： 收集2013年1月至2016年12月的204例肿瘤FFPE标本，包括100例非小细胞肺癌(浙江大学医学院附属第一医院)和104例结直肠癌(四川大学华西医院)。采用相同的病例，一次提取DNA后，相同DNA的量采用相同试剂盒建库，质控合格后分别在Miseq和Ion Torrent PGM平台测序。使用扩增阻滞突变系统(ARMS)PCR法以及Sanger测序对双平台检测不一致的突变进行验证。 结果： 共纳入204个FFPE标本，两个平台共同分析了197个标本。发现Miseq平台测序标本产出的reads数目高于PGM平台(中位数：391 634比298 030，P<0.01)。与人类参照基因组进行比对，结果显示Miseq平台测序标本的比对率高于PGM平台(中位数100.0%比99.7%，P<0.01)。Miseq平台测序标本的中位测序深度高于PGM平台(中位数853×比698×，P<0.01)。两个平台共检测到236个突变，其中221个突变被两者同时检出(一致率93.6%)。Miseq平台比PGM多检出11个突变，而PGM平台比Miseq多检出4个突变。经ARMS－PCR方法及Sanger测序验证，Miseq平台对于低频突变的检测结果更为可靠，两者不一致的主要原因是部分假阴性是由变异频率低于试剂盒报告范围(5%)及石蜡标本保存时间较长导致。 结论： 与PGM平台相比，Miseq平台测序在标本产生的reads数目、比对结果以及覆盖深度方面均表现出了更高的测序质量，且检测结果更为可靠。在临床实践中，应根据标本量和实际的通量需求来选择合适的平台辅助肿瘤分子检测。.

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