Hi,
I am working with microarray data and its experimental design is a bit strange. I have 20 samples (expression at 5 time points, at 1 hr with 3 replicates, 2 hr with 4 replicates, 3 hr with 3 replicates, 6hr with 6 replicates and 10hr with 4 replicates).
I want to find the DEGs at each time point vs rest of time points.
For instance, DEGs at 1 hr vs rest of all time points (2, 3, 6, 10)
DEGs at 2 hr vs rest of all time points (1, 3, 6, 10)
DEGs at 3 hr vs rest of all time points (1, 2, 6, 10) etc..
My design matrix is as follows:
xx <- c(paste0(rep("1hr", 3), sep = ":", paste0(rep("rep", 3), 1:3)), paste0(rep("2hr", 4), sep = ":", paste0(rep("rep", 4), 1:4)), paste0(rep("3hr", 3), sep = ":", paste0(rep("rep", 3), 1:3)), paste0(rep("6hr", 6), sep = ":", paste0(rep("rep", 6), 1:6)), paste0(rep("10hr", 4), sep = ":", paste0(rep("rep", 4), 1:4)))
design <- cbind(complete=1, hr1=c(rep(1, 3), rep(0, 17)), hr2=c(rep(0, 3), rep(1, 4), rep(0, 13)), hr3=c(rep(0, 7), rep(1, 3), rep(0, 10)), hr6=c(rep(0, 10), rep(1, 6), rep(0, 4)), hr10=c(rep(0, 16), rep(1, 4)))
rownames(design) <- xx
My design matrix looks like this:
complete hr1 hr2 hr3 hr6 hr10
1hr:rep1 1 1 0 0 0 0
1hr:rep2 1 1 0 0 0 0
1hr:rep3 1 1 0 0 0 0
2hr:rep1 1 0 1 0 0 0
2hr:rep2 1 0 1 0 0 0
2hr:rep3 1 0 1 0 0 0
2hr:rep4 1 0 1 0 0 0
3hr:rep1 1 0 0 1 0 0
3hr:rep2 1 0 0 1 0 0
3hr:rep3 1 0 0 1 0 0
6hr:rep1 1 0 0 0 1 0
6hr:rep2 1 0 0 0 1 0
6hr:rep3 1 0 0 0 1 0
6hr:rep4 1 0 0 0 1 0
6hr:rep5 1 0 0 0 1 0
6hr:rep6 1 0 0 0 1 0
10hr:rep1 1 0 0 0 0 1
10hr:rep2 1 0 0 0 0 1
10hr:rep3 1 0 0 0 0 1
10hr:rep4 1 0 0 0 0 1
Is this design matrix correct as per my DEGs comparison?
Moreover, I am getting this warning message: " Coefficients not estimable: hr10
Warning message:
Partial NA coefficients for 20214 probe(s) ".
Can anyone help me what's wrong with my design matrix?
Thank you.
> sessionInfo()
R version 3.3.1 (2016-06-21)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.10.5 (Yosemite)
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] Matrix_1.2-6 matrixStats_0.50.2 ggplot2_2.1.0 dplyr_0.5.0 readxl_0.1.1
[6] limma_3.28.15
loaded via a namespace (and not attached):
[1] Rcpp_0.12.6 lattice_0.20-33 digest_0.6.9 assertthat_0.1 grid_3.3.1
[6] R6_2.1.2 plyr_1.8.4 gtable_0.2.0 DBI_0.4-1 magrittr_1.5
[11] scales_0.4.0 lazyeval_0.2.0 labeling_0.3 tools_3.3.1 munsell_0.4.3
[16] colorspace_1.2-6 tibble_1.1
Thank you Aaron. You made it clear. Another question, in the contrasts division by 4 is because of comparing 1 group vs 4 groups?
Yes, you want to compare to the average of the four groups.
Thank you Aaron.