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Parallel factor ChIP provides essential internal control for quantitative differential ChIP-seq.

Published version
Peer-reviewed

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Authors

Guertin, Michael J 
Cullen, Amy E 
Holding, Andrew N 

Abstract

A key challenge in quantitative ChIP combined with high-throughput sequencing (ChIP-seq) is the normalization of data in the presence of genome-wide changes in occupancy. Analysis-based normalization 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 (TF) binding, these assumptions do not hold true. The challenges in normalization are confounded by experimental variability during sample preparation, processing and recovery. We present a novel normalization strategy utilizing an internal standard of unchanged peaks for reference. Our method can be readily applied to monitor genome-wide changes by ChIP-seq that are otherwise lost or misrepresented through analytical normalization. We compare our approach to normalization by total read depth and two alternative methods that utilize external experimental controls to study TF binding. We successfully resolve the key challenges in quantitative ChIP-seq analysis and demonstrate its application by monitoring the loss of Estrogen Receptor-alpha (ER) binding upon fulvestrant treatment, ER binding in response to estrodiol, ER mediated change in H4K12 acetylation and profiling ER binding in patient-derived xenographs. This is supported by an adaptable pipeline to normalize and quantify differential TF binding genome-wide and generate metrics for differential binding at individual sites.

Description

Keywords

Animals, Antibodies, CCCTC-Binding Factor, Chromatin Immunoprecipitation, Drosophila melanogaster, Estrogen Receptor alpha, High-Throughput Nucleotide Sequencing, Histones, Humans, MCF-7 Cells, Mice, Reference Standards, Sequence Analysis, DNA

Journal Title

Nucleic Acids Res

Conference Name

Journal ISSN

0305-1048
1362-4962

Volume Title

46

Publisher

Oxford University Press (OUP)
Sponsorship
Cancer Research UK (C14303/A17197)
Breast Cancer Campaign (2012NovemberPR042)
Cancer Research UK (A19274)