Normalized Affymetrix expression data are biased by G-quadruplex formation. / Shanahan, Hugh P.; Memon, Farhat N.; Upton, Graham J.G.; Harrison, Andrew P.

In: Nucleic Acids Research, Vol. 40, No. 8, 01.04.2012, p. 3307-3315.

Research output: Contribution to journalArticle

Published

Abstract

Probes with runs of 4 or more guanines (G-stacks) in their sequences can exhibit a level of hybridisation that is unrelated to the expression levels of the mRNA that they are intended to measure. This is most likely caused by the formation of G-quadruplexes, where inter-probe guanines form Hoogsteen hydrogen bonds, which probes with G-stacks are capable of forming.

We demonstrate that for a specific microarray data set using the Human HG_U133A Affymetrix GeneChip and RMA normalisation there is significant bias in the expression levels, the fold change and the correlations between expression levels. These effects grow more pronounced as the number of G-stack probes in a probe set increases. Approximately 14% of the probe sets are directly affected. The analysis was repeated for a number of other normalisation pipelines and two, FARMS and PLIER, minimised the bias to some extent.
We estimate that approximately 15% of the data sets deposited in the GEO database are susceptible to the effect.

The inclusion of G-stack probes in the affected data sets can bias key parameters used in the selection and clustering of genes. The elimination
of these probes from any analysis in such affected data sets outweighs the increase of noise in the signal.
Original languageEnglish
Pages (from-to)3307-3315
Number of pages9
JournalNucleic Acids Research
Volume40
Issue number8
Early online date22 Dec 2011
DOIs
Publication statusPublished - 1 Apr 2012
This open access research output is licenced under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

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