[BioC] ChIP-chip sequence bias not removed

[zacher at lmb.uni-muenchen.de]

Dear Edwin,

as I guess from inspecting your code chunck, you did not substract a reference experiment from the IP. To eliminate the sequence-dependent bias you either need to substract a reference experiment (like Mock-IP or genomic input) or apply a sepcific normalization method like MAT, which is designed for this purpose. If you have a reference experiment I absolutely recommend to use this instead of MAT, as it perfoms a lot better in my experience.
Best regards,

Bendikt

Edwin Groot <edwin.groot at biologie.uni-freiburg.de> schrieb :

> Hello all,
> I am analyzing Affymetrix AtTile1F ChIP-chip data from GEO to compare
> the localization of different histone modifications in Arabidopsis. The
> goal is to query a genomic region for relative enrichment of the
> different histone modifications.
> After trying several normalization methods in Starr, I get good MA
> plots, densities and histograms, but neither the GC-bias, nor the
> base-position bias is changed by any normalization method. The vignette
> data, in contrast, shows great improvement in the bias problems. Have I
> missed something? Should I worry about this?
> I have so far tried loess, vsn, quantile and rankpercentile through
> Starr.
> 
> Thanks,
> Edwin
> -- 
> Here is sample code for one of the normalization methods:
> > library(Starr)
> > library(geneplotter)
> > library(vsn)
> > AtTile1F <- readBpmap("GPL1979.bpmap")
> #Only the + strand is represented for all chromosomes
> > summary(AtTile1F$"At:TIGRv5;chr4"$strand)
> > cels <- c("h3k27me301.CEL", "h3k27me303.CEL",
> "h3k27me302.CEL",
> "h3k27me304.CEL", "input01.CEL", "input03.CEL",
> "input02.CEL",
> "input04.CEL")
> > names <- c("k27me301", "k27me302",
> "k27me303", "k27me304", "input01",
> "input02", "input03", "input04")
> > type <- c("IP", "IP", "IP",
> "IP", "INPUT", "INPUT", "INPUT",
> "INPUT")
> > k27me3 <- readCelFile(AtTile1F, cels, names, type, featureData=TRUE,
> log.it=TRUE)
> #Normalize
> > k27me3_loess <- normalize.Probes(k27me3, method = "loess")
> #QC
> #Try only one pair of IP and control.
> > ips <- c(TRUE,FALSE,FALSE,FALSE,FALSE,FALSE,FALSE,FALSE)
> > controls <- c(FALSE,FALSE,FALSE,FALSE,TRUE,FALSE,FALSE,FALSE)
> > plotMA(k27me3, ip = ips, control = controls)
> #There is a negative deviation down to -1.5 LFC
> > plotMA(k27me3_loess, ip = ips, control = controls)
> #The MA is straight, except for a slight negative bias at highest
> intensity.
> > plotGCbias(exprs(k27me3)[, 1], featureData(k27me3)$seq,
> main=paste(sampleNames(k27me3)[1],"GC Bias Before Normalization"))
> #The GC bias increases linearly with base position.
> > plotGCbias(exprs(k27me3_loess)[, 1], featureData(k27me3_loess)$seq,
> main=paste(sampleNames(k27me3_loess)[1],"GC Bias After Loess
> Normalization"))
> #Same rise (-2 to +2) with base position as Before Normalization.
> -- 
> > sessionInfo()
> R version 2.11.1 (2010-05-31) 
> x86_64-pc-linux-gnu 
> 
> locale:
>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
>  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
>  [5] LC_MONETARY=C              LC_MESSAGES=en_US.UTF-8   
>  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
>  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
> 
> attached base packages:
> [1] grid      stats     graphics  grDevices datasets  utils     methods
>  
> [8] base     
> 
> other attached packages:
>  [1] vsn_3.16.0           geneplotter_1.26.0   annotate_1.26.0     
>  [4] AnnotationDbi_1.10.1 Starr_1.4.4          affxparser_1.20.0   
>  [7] affy_1.26.1          Ringo_1.12.0         Matrix_0.999375-40  
> [10] lattice_0.18-8       limma_3.4.3          RColorBrewer_1.0-2  
> [13] Biobase_2.8.0       
> 
> loaded via a namespace (and not attached):
>  [1] affyio_1.16.0         DBI_0.2-5             genefilter_1.30.0    
>  [4] MASS_7.3-6            preprocessCore_1.10.0 pspline_1.0-14       
>  [7] RSQLite_0.9-1         splines_2.11.1        survival_2.35-8      
> [10] xtable_1.5-6         
> 
> Dr. Edwin Groot, postdoctoral associate
> AG Laux
> Institut fuer Biologie III
> Schaenzlestr. 1
> 79104 Freiburg, Deutschland
> +49 761-2032945
> 
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