Wu Chunlei, Zhao Haitao, Baggerly Keith, Carta Roberto, Zhang Li
Genomic Institute of Novartis Research Foundation, 10675 John Jay Hopkins Dr, San Diego, CA 92121, USA.
Bioinformatics. 2007 Oct 1;23(19):2566-72. doi: 10.1093/bioinformatics/btm271. Epub 2007 May 30.
In microarray experiments, probe design is critical to the specific and accurate measurement of target concentrations. Current designs select suitable probes through in silico scanning of transcriptome/genome based on first principles. However, due to lack of tools, the observed microarray data have not been used to assess the performance of individual probes to provide feedback to improve future designs.
In this study, we describe a probe performance assessment method based on the concordance of the observed signals from probes that share common targets. Using this method, we found that probes containing multiple guanines in a row (G-stacks) have abnormal binding behavior compared with other probes, both in gene expression assays and genotyping assays using Affymetrix microarrays. These probes are less likely to covary with other probes that interrogate the same genes. Moreover, we found that these probes are much more likely to produce outliers when fitting the observed signals according to the positional dependent nearest neighbor model, which gives reasonable estimates of binding affinity for most other probes. These results suggest that probes containing G-stacks tend to have increased cross hybridization signals and reduced target-specific hybridization signals, presumably due to multiplex binding forming G-quartet structures. Our findings are expected to be useful in microarray design and data analysis.
在微阵列实验中，探针设计对于靶标浓度的特异性和准确测量至关重要。当前的设计基于第一原理通过对转录组/基因组进行计算机扫描来选择合适的探针。然而，由于缺乏相关工具，所观察到的微阵列数据尚未用于评估单个探针的性能，以提供反馈来改进未来的设计。
在本研究中，我们描述了一种基于共享共同靶标的探针所观察到的信号一致性的探针性能评估方法。使用这种方法，我们发现在使用Affymetrix微阵列进行的基因表达测定和基因分型测定中，与其他探针相比，连续含有多个鸟嘌呤（G-堆叠）的探针具有异常的结合行为。这些探针与询问相同基因的其他探针共变的可能性较小。此外，我们发现，当根据位置依赖的最近邻模型拟合观察到的信号时，这些探针更有可能产生异常值，而该模型能对大多数其他探针的结合亲和力给出合理估计。这些结果表明，含有G-堆叠的探针往往具有增加的交叉杂交信号和降低的靶标特异性杂交信号，推测是由于形成G-四联体结构的多重结合所致。我们的研究结果有望在微阵列设计和数据分析中发挥作用。
