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suparna mitra
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290
@suparna-mitra-5328
Last seen 10.3 years ago
Hello Group,
I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC.
Previously i was using different softwares for this. And this is my
first
try with Bioconductor for big experiment. So thought to get some
advice in
the beginning.
I have Three groups of patient: (In-vivo)
A-Acute reaction. Patient taking a drug X develops reaction.
R-recovered (6 weeks after acute reaction-not longer taking drug X).
T-Tolerant. Patient on X and tolerating treatment.
Now in in-vitro study we used another constant Y
RXY recovered and challenged with X+Y
RY recovered challenged with only Y. RXY vs RY are to exclude
effects by
Y.
TXY tolerant and challenged with X+Y,
TY tolerant challenged with only Y. TXY vs TY are to exclude effects
by Y.
No I want to check the cross relation and effects A vs R, RvsT and Avs
T
and differentially expressed genes for each comparison. And the same
in
invitro. There are not same patients in different groups, thus I think
I
want to apply unpaired-t test.
This is what I tried:
> sessionInfo()
R version 2.15.1 (2012-06-22)
Platform: i386-apple-darwin9.8.0/i386 (32-bit)
locale:
[1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] statmod_1.4.15 limma_3.12.1
annotate_1.34.1 hugene10stprobeset.db_8.0.1
org.Hs.eg.db_2.7.1
[6] BiocInstaller_1.4.7 affycoretools_1.28.0
KEGG.db_2.7.1
GO.db_2.7.1 AnnotationDbi_1.18.1
[11] affy_1.34.0 Biobase_2.16.0
BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0
RSQLite_0.11.1
[16] DBI_0.2-5 oligo_1.20.4
oligoClasses_1.18.0
rmaOligoinvivo = oligo::rma(InVivodat1)
Background correcting
Normalizing
Calculating Expression
> rmaOligoinvitro = oligo::rma(InVitrodat1)
Background correcting
Normalizing
Calculating Expression
> maplot(rmaOligoinvivo)
> maplot(rmaOligoinvitro)
> InVivoTargets
FileName Treatment
1 MC1 A
2 MC2 A
3 MC3 A
4 MC4 A
5 MC5 A
6 MC6 A
7 MC7 R
8 MC8 R
9 MC9 R
10 MC10 R
11 MC11 R
12 MC12 R
13 MC13 T
14 MC14 T
15 MC15 T
16 MC16 T
17 MC17 T
18 MC18 T
>
InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work-
related/Micro_RawData/InVitroTargets.txt")
> InVitroTargets
FileName Treatment Batch CD4
1 MC19 RY 1 High
2 MC20 TY 1 Low
3 MC21 RY 2 High
4 MC22 TY 2 High
5 MC23 TY 2 Low
6 MC24 RY 2 High
7 MC25 TXY 1 Low
8 MC26 RXY 1 High
9 MC27 RXY 2 Low
10 MC28 TXY 2 High
11 MC29 RXY 2 High
12 MC30 TXY 2 High
f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T"))
design.invivo <- model.matrix(~0 + f.invivo)
>
> colnames(design.invivo) <- c("A", "R", "T")
> fit.invivo <- lmFit(rmaOligoinvivo, design.invivo)
> contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels =
design.invivo)
> fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo)
> fit2.invivo <-eBayes(fit2.invivo)
> topTable(fit2.invivo, coef = 1, adjust = "fdr")
ID logFC AveExpr t P.Value adj.P.Val
B
8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013
-2.023533
9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013
-2.023533
18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013
-2.023533
19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013
-2.023533
3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974
-2.323922
19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796
-2.454618
18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796
-2.607991
3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796
-2.685960
6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796
-2.711203
15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796
-2.800385
I am progressing in a right way? Further I want to perform unpaired t
test
for comparing AvsT and so on. Any help will be really great.
Thanks a lot ,
Mitra.
--
Dr. Suparna Mitra
Wolfson Centre for Personalised Medicine
Department of Molecular and Clinical Pharmacology
Institute of Translational Medicine University of Liverpool
Block A: Waterhouse Buildings, L69 3GL Liverpool
Tel. +44 (0)151 795 5394, Internal ext: 55394
M: +44 (0) 7511387895
Email id: smitra@liverpool.ac.uk
Alternative Email id: suparna.mitra.sm@gmail.com
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