A white-box approach to microarray probe response characterization: the BaFL pipeline

Microarrays depend on appropriate probe design to deliver the promise of accurate genome-wide measurement. Probe design, ideally, produces a unique probe-target match with homogeneous duplex stability over the complete set of probes. Much of microarray pre-processing is concerned with adjusting for non-ideal probes that do not report target concentration accurately. Cross-hybridizing probes (non-unique), probe composition and structure, as well as platform effects such as instrument limitations, have been shown to affect the interpretation of signal. Data cleansing pipelines seldom filter specifically for these constraints, relying instead on general statistical tests to remove the most variable probes from the samples in a study. This adjusts probes contributing to ProbeSet (gene) values in a study-specific manner. We refer to the complete set of factors as biologically applied filter levels (BaFL) and have assembled an analysis pipeline for managing them consistently. The pipeline and associated experiments reported here examine the outcome of comprehensively excluding probes affected by known factors on inter-experiment target behavior consistency.

We present here a 'white box' probe filtering and intensity transformation protocol that incorporates currently understood factors affecting probe and target interactions; the method has been tested on data from the Affymetrix human GeneChip HG-U95Av2, using two independent datasets from studies of a complex lung adenocarcinoma phenotype. The protocol incorporates probe-specific effects from SNPs, cross-hybridization and low heteroduplex affinity, as well as effects from scanner sensitivity, sample batches, and includes simple statistical tests for identifying unresolved biological factors leading to sample variability. Subsequent to filtering for these factors, the consistency and reliability of the remaining measurements is shown to be markedly improved.

The data cleansing protocol yields reproducible estimates of a given probe or ProbeSet's (gene's) relative expression that translates across datasets, allowing for credible cross-experiment comparisons. We provide supporting evidence for the validity of removing several large classes of probes, and for our approaches for removing outlying samples. The resulting expression profiles demonstrate consistency across the two independent datasets. Finally, we demonstrate that, given an appropriate sampling pool, the method enhances the t-test's statistical power to discriminate significantly different means over sample classes.