Novel Quantitative ChIP-seq Methods Measure Absolute Fold-Change in ER Binding Upon Fulvestrant Treatment

A key challenge in quantitative ChIP-seq is the normalisation of data in the presence of genome-wide changes in occupancy. Analysis-based normalisation methods were developed for transcriptomic data and these are dependent on the underlying assumption that total transcription does not change between conditions. For genome-wide changes in transcription factor binding, these assumptions do not hold true. Misapplication of these methods to ChIP-seq data results in the suppression of the biological signal or erroneous measurement of differential occupancy. The challenges in normalisation are confounded by experimental variability during sample preparation and processing. Current experimental methodologies do not fully control for variables that influence DNA recovery. We present a novel normalisation strategy utilising an internal standard of unchanged peaks for reference. We compare our approach to normalisation by total read depth and two alternative methods that utilise external controls to study transcription factor binding. We successfully resolve the key challenges in quantitative ChIP-seq analysis and demonstrate its application by monitoring the changes in ∗To whom correspondence should be addressed. Tel: +44 1223 769500; Fax: +44 1223 769510; Email: [email protected] Estrogen Receptor-alpha (ER) binding upon fulvestrantmediated degradation of ER, which compromises ER binding genome-wide. Additionally, we developed an adaptable pipeline to normalise and quantify differential transcription factor binding genome-wide and generate metrics for differential binding at individual sites.