A study of accuracy and precision in oligonucleotide arrays: extracting more signal at large concentrations.
BACKGROUND
Despite the success and popularity of oligonucleotide arrays as a high-throughput technique for measuring mRNA expression levels, quantitative calibration studies have until now been limited. The main reason is that suitable data was not available. However, calibration data recently produced by Affymetrix now permits detailed studies of the intensity dependent sensitivity. Given a certain transcript concentration, it is of particular interest to know whether current analysis methods are capable of detecting differential expression ratios of 2 or higher.


RESULTS
Using the calibration data, we demonstrate that while current techniques are capable of detecting changes in the low to mid concentration range, the situation is noticeably worse for high concentrations. In this regime, expression changes as large as 4 fold are severely biased, and changes of 2 are often undetectable. Such effects are mainly the consequence of the sequence specific binding properties of probes, and not the result of optical saturation in the fluorescence measurements. GeneChips are manufactured such that each transcript is probed by a set of sequences with a wide affinity range. We show that this property can be used to design a method capable of reducing the high intensity bias. The idea behind our methods is to transfer the weight of a measurement to a subset of probes with optimal linear response at a given concentration, which can be achieved using local embedding techniques.


BACKGROUND
Program source code will be sent electronically upon request.
Naef
Felix
F
Center for Studies in Physics and Biology, Rockefeller University, 1230 York Avenue, NY 10021, USA. felix@funes.rockefeller.edu
Socci
Nicholas D
ND
Magnasco
Marcelo
M
D000465:Algorithms; D002138:Calibration; D019544:Equipment Failure Analysis / Q000379:methods*; D015335:Molecular Probes; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D025341:Principal Component Analysis; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013269:Stochastic Processes; D015003:Yeasts / Q000235:genetics
D015335:Molecular Probes