Abstract
BackgroundAlthough oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches.ResultsIn this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA) will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay.ConclusionsBy integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.
Highlights
Oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers
Algorithm In simulations of microarray hybridization, the extreme computational burden of the Direct Method and the memory burden of the Reaction Method may be alleviated by judicious storage and summation of the terms in Eq 6
We have developed an algorithm for the stochastic simulation of exceptionally large and complex probe-cDNA hybridization reaction networks that underlie microarray assays
Summary
Oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. The hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. There are several high throughput methods of quantifying changes in gene expression including oligonucleotide microarrays, quantitative realtime PCR (qPCR) and " generation sequencing" Generation sequencing is a promising alternative to microarrays for genome-scale expression profiling, and exhibit more sensitivity in the low-expression limit [2,3], microarray technology is substantially less expensive and the resulting data sets require much less information processing. Probes may be attached to (or synthesized from) the slide surface via (1) maskdependent [4,5,6,7,8,9] or maskless photolithographic DNA synthesis technology [10,11], or (2) robotic printing of PCR products or synthetic oligomers [12]. The first two of these methods yield oligonucleotide arrays (e.g. Affymetrix (page number not for citation purposes)
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