InterpolatedXY: a two-step strategy to normalize DNA methylation microarray data avoiding sex bias

Publication type

Journal Article


Publication date

August 15, 2022



Data normalization is an essential step to reduce technical variation within and between arrays. Due to the different karyotypes and the effects of X chromosome inactivation, females and males exhibit distinct methylation patterns on sex chromosomes; thus, it poses a significant challenge to normalize sex chromosome data without introducing bias. Currently, existing methods do not provide unbiased solutions to normalize sex chromosome data, usually, they just process autosomal and sex chromosomes indiscriminately.

Here, we demonstrate that ignoring this sex difference will lead to introducing artificial sex bias, especially for thousands of autosomal CpGs. We present a novel two-step strategy (interpolatedXY) to address this issue, which is applicable to all quantile-based normalization methods. By this new strategy, the autosomal CpGs are first normalized independently by conventional methods, such as funnorm or dasen; then the corrected methylation values of sex chromosome-linked CpGs are estimated as the weighted average of their nearest neighbors on autosomes. The proposed two-step strategy can also be applied to other non-quantile-based normalization methods, as well as other array-based data types. Moreover, we propose a useful concept: the sex explained fraction of variance, to quantitatively measure the normalization effect.
Availability and implementation:

The proposed methods are available by calling the function ‘adjustedDasen’ or ‘adjustedFunnorm’ in the latest wateRmelon package (, with methods compatible with all the major workflows, including minfi.
Supplementary information:

Supplementary data are available at Bioinformatics online.

Published in


Volume and page numbers

Volume: 38 , p.3 -3






Open Access

© The Author(s) 2022. Published by Oxford University Press

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact



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