[BioC] limma - background correct method=none

[Helen Cattan]

Hi,
 
I have been looking at backgroundCorrect, method=none in limma and
compared this to files I have manually altered so that the background
values were zero, without using the backgroundCorrect method. I thought
that these would produce the same results but they were very different -
could anyone explain why to me please? Code is below.
Thanks,
Helen
 
> library(limma)
> files=dir(pattern="*\\.gpr")
> RG=read.maimages(files, columns=list(Rf="F635 Median", Gf="F532
Median", Rb="B635 
> names(RG)
> RG$genes=readGAL()
> RG$printer=getLayout(RG$genes)
> samples=read.table("sampleinformationa.txt", header=TRUE, sep="\t",
as.is=TRUE)
> samples
> spottypes=readSpotTypes()
> RG$genes$Status=controlStatus(spottypes, RG)
> MA1=normalizeWithinArrays(RG)
> MA2=normalizeBetweenArrays(MA1)
> design=c(1,-1)
> cor=dupcor.series(MA2$M, design, ndups=2, spacing=1)
> cor$cor
> fit=gls.series(MA2$M,design,ndups=2,correlation=0.7628949)
> eb=ebayes(fit)
> genenames=uniquegenelist(RG$genes, ndups=2) 
> ord=order(eb$lods, decreasing=TRUE)
> toptable(number=30,genelist=genenames,fit=fit,eb=eb,adjust="fdr")
     Block Row Column         ID
Name
2986    10  24     19     209274
H63351:Hs.203509::::3:211600
5981    20  24      9     No_seq                    :Data not
found:::::212310
8083    27  24     13     No_seq                    :Data not
found:::::211255
2013     7  18     17    3846240     BE617901:In multiple
clusters::::3:152735
7492    25  25      7     No_seq                    :Data not
found:::::223083
     Status        M        t     P.Value        B
2986   cDNA 2.032293 21.15504 0.001026994 8.082774
5981   cDNA 1.946782 17.10056 0.001115381 6.944303
8083   cDNA 1.957218 16.81269 0.001115381 6.847414
2013   cDNA 1.659813 15.65550 0.001115381 6.431781
7492   cDNA 1.739500 15.31887 0.001115381 6.302453
> top30=ord[1:30]
> plot(fit$coef,eb$lods,xlab="Log2 Fold Change", ylab="Log
Odds",pch=16,cex=0.1)
 
 and then included RG=backgroundCorrect(RG, method="none") without
manually altering the files, before the normalizations and got the
following top table.
The MA plots were also very different between the two tests.
 
Block Row Column         ID
Name
4062    14  14     11     130050             R11620:Data not
found::::0:130050
1033     4  12      1     124297             R02202:Data not
found::::0:124297
1551     6   5      5    5195930           BI754638:Data not
found::::10:39204
6900    23  25     23     214572       H73225:In multiple
clusters::::0:214572
5131    18   3     13        N/A           BQ025821:Data not
found::::10:33091
     Status        M         t   P.Value           B
4062   cDNA 2.421487 11.601562 0.1968434 -0.08869236
1033   cDNA 1.681192  8.298373 0.1968434 -0.58215799
1551   cDNA 1.904751  8.293908 0.1968434 -0.58312702
6900   cDNA 2.094721  8.171346 0.1968434 -0.61016473
5131   cDNA 2.314669  7.761475 0.1968434 -0.70707369
 
 

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