title: Is this the right normalization? A diagnostic tool for ChIP-seq normalization.
abstract: BACKGROUND
  Chip-seq experiments are becoming a standard approach for genome-wide profiling protein-DNA interactions, such as detecting transcription factor binding sites, histone modification marks and RNA Polymerase II occupancy. However, when comparing a ChIP sample versus a control sample, such as Input DNA, normalization procedures have to be applied in order to remove experimental source of biases. Despite the substantial impact that the choice of the normalization method can have on the results of a ChIP-seq data analysis, their assessment is not fully explored in the literature. In particular, there are no diagnostic tools that show whether the applied normalization is indeed appropriate for the data being analyzed.
  
  
  RESULTS
  In this work we propose a novel diagnostic tool to examine the appropriateness of the estimated normalization procedure. By plotting the empirical densities of log relative risks in bins of equal read count, along with the estimated normalization constant, after logarithmic transformation, the researcher is able to assess the appropriateness of the estimated normalization constant. We use the diagnostic plot to evaluate the appropriateness of the estimates obtained by CisGenome, NCIS and CCAT on several real data examples. Moreover, we show the impact that the choice of the normalization constant can have on standard tools for peak calling such as MACS or SICER. Finally, we propose a novel procedure for controlling the FDR using sample swapping. This procedure makes use of the estimated normalization constant in order to gain power over the naive choice of constant (used in MACS and SICER), which is the ratio of the total number of reads in the ChIP and Input samples.
  
  
  CONCLUSIONS
  Linear normalization approaches aim to estimate a scale factor, r, to adjust for different sequencing depths when comparing ChIP versus Input samples. The estimated scaling factor can easily be incorporated in many peak caller algorithms to improve the accuracy of the peak identification. The diagnostic plot proposed in this paper can be used to assess how adequate ChIP/Input normalization constants are, and thus it allows the user to choose the most adequate estimate for the analysis.
authors.affiliation: Istituto per le Applicazioni del Calcolo "Mauro Picone", Via Pietro Castellino, 111, Naples, 80131, Italy. c.angelini@iac.cnr.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Angelini
authors.affiliation: Department of Statistics and Operations Research Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel. ruheller@post.tau.ac.il.
authors.forename: Ruth
authors.initials: R
authors.lastname: Heller
authors.affiliation: Department of Statistics and Operations Research Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel. spirita123@gmail.com.
authors.forename: Rita
authors.initials: R
authors.lastname: Volkinshtein
authors.affiliation: Department of Statistics and Operations Research Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel. yekutiel@post.tau.ac.il.
authors.forename: Daniel
authors.initials: D
authors.lastname: Yekutieli
mesh_terms: D000465:Algorithms; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003627:Data Interpretation, Statistical; D016681:Genome, Fungal; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006657:Histones / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II