Jiang R R, Wang Y J, Teng X D, Xiao L, Bu H, Ye F
Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhonghua Bing Li Xue Za Zhi. 2018 Aug 8;47(8):591-596. doi: 10.3760/cma.j.issn.0529-5807.2018.08.005.
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. 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. 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 . 298 030, <0.01). Alignment with human reference genome showed that the mapping rate of Miseq platform was higher than PGM platform (median 100.0% . 99.7%, <0.01). The median sequence depth of samples on Miseq was higher than PGM platform (median 853× . 698×, <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. 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.
比较Miseq和Ion Torrent PGM平台的性能以及用于福尔马林固定石蜡包埋（FFPE）样本的新一代测序（NGS）技术的文库构建方法。从2013年1月至2016年12月，回顾性选取了浙江大学第一附属医院的100例非小细胞肺癌和四川大学华西医院的104例结直肠癌，共204例FFPE癌症样本。使用相同的样本提取DNA，用相同的试剂盒以相同量的DNA进行文库构建，并在通过质量控制后分别在Miseq和Ion Torrent PGM上进行测序。两个平台之间任何不一致的突变均通过扩增阻滞突变系统-聚合酶链反应（ARMS-PCR）方法和桑格测序进行验证。共纳入204例FFPE样本，两个平台成功分析了197例样本。样本在Miseq平台测序产生的读数数量高于PGM平台（中位数391634对298030，<0.01）。与人类参考基因组比对显示，Miseq平台的映射率高于PGM平台（中位数100.0%对99.7%，<0.01）。样本在Miseq上的中位序列深度高于PGM平台（中位数853×对698×，<0.01）。两个平台共检测到236个突变，其中221个在两个平台上均被检测到，一致性为93.6%。Miseq平台检测到11个PGM平台未检测到的突变，而PGM平台检测到4个Miseq平台未检测到的更多突变。经ARMS-PCR和桑格测序验证，Miseq平台对低频突变更可靠。两个平台之间不一致突变的主要原因是未检测平台上的突变频率低于突变报告范围（5%）以及FFPE样本储存时间较长。与PGM相比，Miseq平台在读数数量、比对结果和覆盖深度方面显示出更高的测序质量，检测结果更可靠。在临床实践中，应根据样本量和实际通量需求选择合适的平台，以辅助肿瘤的分子特征分析。
