No DEG detected in microarray data

Question

[Aki]

hello I'm trying to analyze microarray data (affymetrix.genechip.hg-u133_plus_2). but my data has no genes with adj.P.Val < 0.05 using limma. Could you tell me what are there some wrong code or my sample is bad? my paired sample (5 sample/control (before) and treatment (after)).

head(data)
CTRL_1 CTRL_2 CTRL_3 CTRL_4 CTRL_5  Treat_1  Treat_2  Treat_3 Treat_3 Treat_4 Treat_5
AFFX-BioB-5_at     3649.6    1649.3    1838.3    2103.8    2848.2  2861.1  1717.9  1819.6  2327.9  2621.5
AFFX-BioB-M_at     4726.6    2108.9    2550.0    3696.8    4226.2  3643.5  2260.9  2578.2  3408.9  4177.5
AFFX-BioB-3_at     3179.6    1490.6    1545.3    2186.2    2613.6  2598.8  1400.5  1653.3  2389.5  2633.4

pairinfo <- factor(rep(1:5,2))
group_list <- c(rep("CTRL",5), rep("Treat",5))
group_list <- factor(group_list,levels = c("CTRL","IR"),ordered = F)
design <- model.matrix(~ pairinfo+group_list)
design_CTRL
 (Intercept) pairinfo2 pairinfo3 pairinfo4 pairinfo5 group_list
1            1         0         0         0         0                 0
2            1         1         0         0         0                 0
3            1         0         1         0         0                 0
4            1         0         0         1         0                 0
5            1         0         0         0         1                 0
6            1         0         0         0         0                 1
7            1         1         0         0         0                 1
8            1         0         1         0         0                 1
9            1         0         0         1         0                 1
10           1         0         0         0         1                 1

fit <- lmFit(data,design)
fit <- eBayes(fit)
DEG <- topTable(fit, adjust = 'BH',coef = 6, number = Inf)
summary(DEG_CTRL)
 logFC                t               P.Value            adj.P.Val            B         
Min.   :-8722.22   Min.   :-12.2441   Min.   :0.0000383   Min.   :0.3105   Min.   :-4.613  
 1st Qu.:  -34.62   1st Qu.: -1.1654   1st Qu.:0.1315141   1st Qu.:0.5260   1st Qu.:-4.608  
 Median :   -2.90   Median : -0.1378   Median :0.3569465   Median :0.7139   Median :-4.595  
 Mean   :   30.27   Mean   : -0.1634   Mean   :0.4074041   Mean   :0.6982   Mean   :-4.587  
 3rd Qu.:   24.41   3rd Qu.:  0.8689   3rd Qu.:0.6618781   3rd Qu.:0.8824   3rd Qu.:-4.572  
 Max.   :15670.46   Max.   : 14.0514   Max.   :1.0000000   Max.   :1.0000   Max.   :-4.511 

[German.M.Demidov]

It may just mean that the treatment has no effect on gene expression

[German.M.Demidov]

also I believe DeSeq2 is better (more powerful) for such small (it is considered quite small) sample sizes

[Michael]

DeSeq is for RNA-seq, this is microarray data. I rather suspect there is something wrong with the experiment design or the formula. But I don't know what the real design is and what the factors are. However, for all factors except group you seemingly have only 2 of 10 samples for value 1. I think you should start with a simpler design, just CTRL vs Treat and see what happens. If the pair_info data denote the same patient, you should rather encode them in a single factor with patient id. Also then a more appropriate model formula would be ~group + group*patient. the more I think about it, I believe it is your model matrix that is the cause.

[cpad0112]

I think it's because you didn't bother check the output from your own code. Try checking the output/group_list posted in your code:

group_list <- c(rep("CTRL",5), rep("Treat",5))
group_list <- factor(group_list,levels = c("CTRL","IR"),ordered = F)

Please always keep analysis simple, when you are testing your code and your data.

Answer 1

[Aki]

Thank you kind guys, I checked my design and I realized that I was trying to compare between patients, not CTRL vs Treat!

I got much of DEG by treatment.