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Old 01-18-2012, 02:20 AM   #1
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Default ChIP-Seq: A strand specific high resolution normalization method for chip-sequencing

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A strand specific high resolution normalization method for chip-sequencing data employing multiple experimental control measurements.

Algorithms Mol Biol. 2012 Jan 16;7(1):2

Authors: Enroth S, Andersson CR, Andersson R, Wadelius C, Gustafsson MG, Komorowski J

Abstract
ABSTRACT: BACKGROUND: High-throughput sequencing is becoming the standard tool for investigating protein-DNA interactions or epigenetic modifications. However, the data generated will always contain noise due to e.g. repetitive regions or non-specific antibody interactions. The noise will appear in the form of a background distribution of reads that must be taken into account in the downstream analysis, for example when detecting enriched regions (peak-calling). Several reported peak-callers can take experimental measurements of background tag distribution into account when analysing a data set. Unfortunately, the background is only used to adjust peak calling and not as a pre-processing step that aims at discerning the signal from the background noise. A normalization procedure that extracts the signal of interest would be of universal use when investigating genomic patterns. RESULTS: We formulated such a normalization method based on linear regression and made a proof-of-concept implementation in R and C++. It was tested on simulated as well as on publicly available ChIP-seq data on binding sites for two transcription factors, MAX and FOXA1 and two control samples, Input and IgG. We applied three different peak-callers to (i) raw (un-normalized) data using statistical background models and (ii) raw data with control samples as background and (iii) normalized data without additional control samples as background. The fraction of called regions containing the expected transcription factor binding motif was largest for the normalized data and evaluation with qPCR data for FOXA1 suggested higher sensitivity and specificity using normalized data over raw data with experimental background. CONCLUSIONS: The proposed method can handle several control samples allowing for correction of multiple sources of bias simultaneously. Our evaluation on both synthetic and experimental data suggests that the method is successful in removing background noise.


PMID: 22248020 [PubMed - as supplied by publisher]



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