桑格测序
融合基因
DNA
生物
分子生物学
连续稀释
核糖核酸
基因
DNA测序
遗传学
医学
病理
替代医学
作者
Hao Peng,Rong Huang,Kui Wang,Cuiyun Wang,Bin Li,Youbing Guo,Meng Li,Dadong Zhang,Dong Hua,Hao Chen,Caifu Chen,Qiyou Xu,Fugen Li,Lei Tian,Jianbing Wu
标识
DOI:10.1016/j.jmoldx.2020.11.005
摘要
RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh-frozen tumors; however, RNA-seq is much more challenging to use with formalin-fixed, paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and six known fusions from five cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra-assay and interassay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which found that this is a false-negative result for the DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, 20 (76.9%) of which were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could complement DNA-based next-generation sequencing assays.
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