Widespread misinterpretable ChIP-seq bias in yeast.
Widespread misinterpretable ChIP-seq bias in yeast.
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DOI：
10.1371/journal.pone.0083506
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Iyer VR
中科院分区：
文献类型：
--
作者：
Park D;Lee Y;Bhupindersingh G;Iyer VR
染色质免疫沉淀测序（ChIP-seq）被广泛用于检测体内感兴趣的蛋白质和DNA之间的全基因组相互作用。在相邻背景区域上显示出强富集的基因座通常被认为是结合位点。对ChIP-seq程序固有的系统性人为因素关注不够，这些人为因素可能会产生蛋白质与某些基因座结合的误导性图像。我们在这里表明，无关的转录因子似乎始终结合到酵母中高度转录基因的基因体。引人注目的是，几种类型的阴性对照实验，包括预期不结合染色质的蛋白质，也显示出基因体内强结合的类似模式。这些假阳性信号在测序平台和免疫沉淀方案以及其他实验室先前发表的数据集中都很明显。我们表明，这些假阳性信号来自高转录率，是固有的ChIP程序，虽然他们加剧了测序文库构建程序。这种表达偏好足够强，以至于像Tup 1这样的已知转录抑制因子可能错误地表现为激活因子。另一种类型的背景偏差源于染色质固有的核小体结构，并且可能使某些因子看起来像是结合了核小体，即使它们没有。我们的分析表明，模拟ChIP样品提供了一个更好的表达偏差的归一化控制，而ChIP输入更适合于核小体周期性偏差。虽然这些控制措施在一定程度上减轻了偏差的影响，但它们无法完全消除偏差。因此，谨慎是有道理的数据，似乎显示各种转录和染色质因子与酵母中的高转录基因的关联解释。
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to detect genome-wide interactions between a protein of interest and DNA in vivo. Loci showing strong enrichment over adjacent background regions are typically considered to be sites of binding. Insufficient attention has been given to systematic artifacts inherent to the ChIP-seq procedure that might generate a misleading picture of protein binding to certain loci. We show here that unrelated transcription factors appear to consistently bind to the gene bodies of highly transcribed genes in yeast. Strikingly, several types of negative control experiments, including a protein that is not expected to bind chromatin, also showed similar patterns of strong binding within gene bodies. These false positive signals were evident across sequencing platforms and immunoprecipitation protocols, as well as in previously published datasets from other labs. We show that these false positive signals derive from high rates of transcription, and are inherent to the ChIP procedure, although they are exacerbated by sequencing library construction procedures. This expression bias is strong enough that a known transcriptional repressor like Tup1 can erroneously appear to be an activator. Another type of background bias stems from the inherent nucleosomal structure of chromatin, and can potentially make it seem like certain factors bind nucleosomes even when they don't. Our analysis suggests that a mock ChIP sample offers a better normalization control for the expression bias, whereas the ChIP input is more appropriate for the nucleosomal periodicity bias. While these controls alleviate the effect of the biases to some extent, they are unable to eliminate it completely. Caution is therefore warranted regarding the interpretation of data that seemingly show the association of various transcription and chromatin factors with highly transcribed genes in yeast.
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DOI：
10.1073/pnas.93.11.5208
发表时间：
1996-05-28
影响因子：
11.1
作者：
Iyer, V;Struhl, K
通讯作者：
Struhl, K
影响因子：
30.5
作者：
Kidder, Benjamin L.;Hu, Gangqing;Zhao, Keji
通讯作者：
Zhao, Keji
影响因子：
9.8
作者：
Li XY;MacArthur S;Bourgon R;Nix D;Pollard DA;Iyer VN;Hechmer A;Simirenko L;Stapleton M;Luengo Hendriks CL;Chu HC;Ogawa N;Inwood W;Sementchenko V;Beaton A;Weiszmann R;Celniker SE;Knowles DW;Gingeras T;Speed TP;Eisen MB;Biggin MD
通讯作者：
Biggin MD
影响因子：
64.5
作者：
Holstege, FCP;Jennings, EG;Young, RA
通讯作者：
Young, RA
影响因子：
3.7
作者：
Fan X;Struhl K
通讯作者：
Struhl K