本次解读2篇文章，内容是对几款基于扩增子测序的融合检测Assay的性能对比，测试的样本类型包括融合已知的FFPE和细胞系、首次测序的冰冻保存癌症组织样本，得到的结果：

• 在都能够检测已知融合的情况下，Assay的检测成本、样本的要求（起始量和质量）更能影响客户对Assay的选择，往往会忽略检测技术本身的优势

• 基于AMP和SPE技术（可检未知融合）的Assay能够检测未知的融合伴侣基因，提升融合基因的检出比例

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文献1^[1]

亮点：测试的样本为冻存的前列腺癌样本，样本先前未测试

Design of the comparison

研究旨在端对端过程比较检测不同类型融合的能力，而不是比较每种assay的各个组成部分。

• 同样的RNA样本在不同Assay上进行了测试
• 比较范围限定在所有assay共有的19个基因范围内，其中ERG、ETV1、ETV4和TMPRSS2被证明参与融合
• 用制造商的检出标准来评估检出变异的重现性

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各试剂盒融合检出情况

• 鉴定出5种融合基因(与 ETS 转录因子相关) (Figure 1): TMPRSS2-ERG (n=12), TMPRSS2-ETV4 (n=1), MALAT1-ETV1 (n=1), SLC45A3-ETV1 (n=1), and SNRPN-ETV1(n=1).

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检出融合的进一步分析

对于融合伴侣和断点已知的融合基因（TMPRSS2-ERG），所有Assay都成功地检测

Table 2: Detailed exon coverage of each assay and the detection of fusions with known partners and known breakpoint (TMPRSS2-ERG) or unknown breakpoint (TMPRSS2-ETV4)

对于融合伴侣已知但断点未知的融合基因TMPRSS2-ETV4，3个Assay能成功检出(其中1个Assay有测序reads，但分析时未能识别出)

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对于融合伴侣和断点未知的融合基因，2个Assay能检出

• OCAv3和AICv3不能检测未知的融合伴侣
• FusionPlex和QIAseq在一侧融合已知的情况下，可以检测未知的partner基因

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文献2^[2]

亮点：与捕获法的试剂盒同时进行了对比；用已知融合的细胞系和FFPE样本进行了评估；用制造商不同版本的软件进行了分析评估

5个试剂盒的对比

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检测结果

• a. 8个细胞系梯度稀释样本中检出的结果；部分融合在特定稀释梯度及对应的软件版本时无法检出
• b. 18例融合已知的FFPE样本检出结果对比；仅有2个Assay能明确检出所有已知融合

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最终意见

• In summary, only the TruSight Tumor 170 Assay (Illumina) detected all fusions analyzed in both cell line and FFPE tissue samples. Further, this assays showed the smallest number of false positive results and used only up to 85 ng of RNA.
• In our study, the Archer FusionPlex Lung Assay (ArcherDX) had the highest RNA quality requirements or failed otherwise. Among the RNA-based assays the QIAseq RNAscan Custom Panel (Qiagen) had the highest number of false positive fusion events.
• Although only 10 ng of RNA were needed, the Oncomine Focus Assay (Thermo Fisher Scientific) was the least adequate assay for our purposes, as seven targetable lung cancer fusion events were not covered by the assay and two fusions were classified as uncertain.
• Also the DNA-based SureSelect XT HS Custom Panel (Agilent) did not detect three fusions and in nine samples the fusion was only detected in one of the two software version. Further, many false positive fusions were detected, which makes the detection difficult in an unknown sample.

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参考资料

1. Qu, X., Yeung, C., Coleman, I., Nelson, P.S. & Fang, M. Comparison of four next generation sequencing platforms for fusion detection: Oncomine by ThermoFisher, AmpliSeq by illumina, FusionPlex by ArcherDX, and QIAseq by QIAGEN. Cancer Genetics 243, 11-18 (2020).
2. Heydt, C., Wölwer, C.B., Velazquez Camacho, O. et al. Detection of gene fusions using targeted next-generation sequencing: a comparative evaluation. BMC Med Genomics 14, 62 (2021).