Simple Microarray Analysis Using R
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Ahmed Moustafa [email protected] @AhmedMoustafa
Slides: http://bit.ly/microarray-analysis-using-R-slides
Script: http://bit.ly/microarray-analysis-using-R-script
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¡ Ho: There is NO difference between expression levels of WT and KO
¡ HA: There is a difference
¡ Two-sided or one-sided test?
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Total RNA
cDNA
Hybridization Affymetrix GeneChip
Data Analysis
HDAC1 wild-type [WT] ESC
HDAC1 knockout [KO] ESC
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http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5583
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GSE5583 - Histogram
data
Frequency
0 10000 20000 30000 40000 50000
020000
40000
60000
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GSE5583 (log2) - Histogram
data2
Frequency
0 5 10 15
02000
4000
6000
8000
10000
12000
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WT.GSM130365
WT.GSM130366
WT.GSM130367
KO.GSM130368
KO.GSM130369
KO.GSM130370
0
5
10
15
GSE5583 - boxplots
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WT
KO
KO.GSM130368
KO.GSM130369
KO.GSM130370
WT.GSM130366
WT.GSM130365
WT.GSM130367
0.070
0.075
0.080
0.085
0.090
GSE5583 - Hierarchical Clustering
hclust (*, "complete")as.dist(1 - cor(data2))
Height
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0 5 10 15
05
1015
GSE5583 - Scatter
WT
KO
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NOT biologically significant (NO change)
Biologically significant (down-regulated in KO)
Biologically significant (up-regulated in KO)
Histogram of fold
fold
Frequency
-6 -4 -2 0 2 4 6
01000
2000
3000
4000
5000
6000
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0.0
0.1
0.2
0.3
0.4
9 12 15 18
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0.0
0.1
0.2
0.3
0.4
8 10 12
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Histogram of -log10(pvalue)
-log10(pvalue)
Frequency
0 1 2 3 4 5 6
02000
4000
6000
NOT statistically significant
Statistically significant differentially expressed
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-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano
fold
-log10(pvalue)
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-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano
fold
-log10(pvalue)
-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano
fold
-log10(pvalue)
Statistically significant
Up-regulated Down-regulated
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42 168 387
fold- change p-value
12,488
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-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano #2
fold
-log10(pvalue)
How about highlighting the up-regulated and down-regulated genes in different colors?
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-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano #3
fold
-log10(pvalue)
-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano #3
fold
-log10(pvalue)
-6 -4 -2 0 2 4 6
01
23
45
GSE5583 - Volcano #3
fold
-log10(pvalue)
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KO.GSM130369
KO.GSM130368
KO.GSM130370
WT.GSM130366
WT.GSM130365
WT.GSM130367
104480_at162349_i_at94027_at94028_f_at101883_s_at93028_at96643_at94085_at97661_at102712_at103244_at104526_at95430_f_at98813_at99472_at162395_r_at100914_at95291_r_at102726_at92331_at95919_at100716_at160172_at92708_at101368_at160943_at94421_r_at101635_f_at98623_g_at92681_at96046_at98621_at103088_at103299_at104700_at93541_at95471_at93691_s_at101550_at98347_at104425_at162405_at
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KO.GSM130369
KO.GSM130368
KO.GSM130370
WT.GSM130366
WT.GSM130365
WT.GSM130367
104480_at162349_i_at94027_at94028_f_at101883_s_at93028_at96643_at94085_at97661_at102712_at103244_at104526_at95430_f_at98813_at99472_at162395_r_at100914_at95291_r_at102726_at92331_at95919_at100716_at160172_at92708_at101368_at160943_at94421_r_at101635_f_at98623_g_at92681_at96046_at98621_at103088_at103299_at104700_at93541_at95471_at93691_s_at101550_at98347_at104425_at162405_at
-1 0 1
Row Z-Score
02
46
8
Color Keyand Histogram
Count
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Functional annotation of the subset of differential expressed genes
http://david.abcc.ncifcrf.gov/
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Thank You!