Dear edgeR users, I am not a statistician nor R programming geek, please forgive me if I ask stupid question. I have RNA-seq data for 2 different genotype of trees with different time points 0hour(control),3hr,24hrs,and 48hrs. Each time point has two replicates. The experiment design like as below: Sample harvested after treatment at Tree H1 Ctrl 3hrs 24hrs 48hrs Tree H2 Ctrl 3hrs 24hrs 48hrs Tree L1 Ctrl 3hrs 24hrs 48hrs Tree L2 Ctrl 3hrs 24hrs 48hrs I would like to study genes that are differentially expressed throughout the time points in these 2 genotype of trees with edgeR. I read from the edgeR user guide, the suitable DE analysis method for my expriment is GLM likelihood ratio test. After read the user guide, I have the RNA-Seq counts in a file like below in order to input into the edgeR package: Ref Tags H1_C H2_C H1_3H H2_3H H1_1D H2_1D H1_2D H2_2D L1_C L2_C L1_3H L2_3H L1_1D L2_1D L1_2D L2_2D AA212259 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 AA216460 1 0 0 1 2 1 6 2 1 1 1 0 5 1 3 1 AA221873 3 0 1 0 3 0 0 2 1 0 1 0 2 1 2 0 AA235900 3 1 6 0 5 4 4 4 7 2 4 3 3 0 8 0 I used the following commands to input the counts file into edgeR: rawdata <- read.delim("file.txt", sep="\t", check.name=FALSE, stringsAsFactor=FALSE) trees <- factor(c("HS","HS","HS","HS","HS","HS","HS","HS","LS","LS","LS","LS"," LS","LS","LS","LS")) treat <- factor(c("C1","C2","3h1","3h2","24h1","24h2","48h1","48h2","C1","C2"," 3h1","3h2","24h1","24h2","48h1","48h2")) I'm not good in R programming, thus, having this file input into the edgeR and assign the factors as well as design-matrix is a challenge. I'm stuck how to tell the edgeR about my design matrix. Could you guys kindly help me on this? Have I input my counts correctly? Please correct me if there is any mistake I have done in my edgeR input. I appreciate very much for your help and time. Have a nice day. Best regards, KJ Lim [[alternative HTML version deleted]]