Systematic analysis of RNAi reports identifies dismal commonality at gene-level and reveals an unprecedented enrichment in pooled shRNA screens.
Systematic analysis of RNAi reports identifies dismal commonality at gene-level and reveals an unprecedented enrichment in pooled shRNA screens.
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DOI：
10.2174/13862073113169990045
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发表时间：
2013-11
影响因子：
1.8
通讯作者：
Djaballah H
中科院分区：
文献类型：
--
作者：
Bhinder B;Djaballah H
RNA干扰（RNAi）为更好地了解基因功能开辟了有希望的途径。尽管许多RNAi筛选报告了基因的鉴定，但很少有（如果有的话）得到进一步的研究和验证。数据差异正在成为RNAi的主要陷阱之一。我们推断，对基于致死率的筛选进行系统分析，因为它们对细胞死亡进行评分，将检查筛选间水平的命中不一致的程度。为此，我们开发了一种方法，用于文献挖掘和重叠分析的几个屏幕使用siRNA和shRNA的口味，并获得了64个基因列表审查的初始列表的7,430个提名的基因。我们进一步进行了比较分析，首先在全球范围内，然后在更严格的条件下进行重新评估。令我们惊讶的是，即使对于PLK 1，也没有任何一个命中重叠，PLK 1在siRNA筛选中成为一个强有力的候选者;但在shRNA筛选中只有少量。此外，EIF 5 B仅在shRNA筛选中成为最常见的命中。一个非常不寻常和前所未有的结果是观察到在shRNA筛选中的6，664个指定基因中有5，269个（~80%）专用于合并格式，引起了对合并筛选的优点的关注，其基于相对消耗来鉴定命中，可能是由于每个细胞的多次整合、数据去卷积或细胞内处理中的不准确性导致脱靶效应。在没有黄金标准的情况下，我们鼓励社区更多地关注RNAi筛选数据分析实践，记住它本质上是组合的，每个基因一个活性siRNA双链体或shRNA发夹不足以获得可信的命中提名。最后，我们还希望谨慎解释合并的shRNA筛选结果。
RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes.