Hello All,

I am applying Voom normalization to RNA-Seq raw Counts data obtained from TCGA. I have constructed a Matrix of ~20000 Rows and 341 Columns with first column being of Gene_id.

I am using Voom() method to normalise the data. I have done the following code.

## Libraries
library(limma)
library(edgeR)

## Matrix File
raw.data <-read.delim("Combined_matrix_340.txt")
attach(raw.data)
names(raw.data)
d <- raw.data[, 2:341]
rownames(d) <- raw.data[, 1]

# Pheno data file
pheno<-read.table("pheno_data_BRCA.txt", header=TRUE, sep="\t")

##To design matrix---
Group<-factor(pheno$Status,levels=levels(pheno$Status))
design<-model.matrix(~0+Group)

##Normalisation 
y <- voom(d,design,plot=TRUE)

colnames(design)

fit <-lmFit(y,design)

##Designing Contrast Matrix for group Differentiation

cont.wt<-makeContrasts("Metastatic-Normal_Control","ERPositive-Normal_Control","PRPositive-Normal_Control","HER2Positive-Normal_Control","ER_PR_HER2_Neg-Normal_Control",levels=design)

fit2 <-contrasts.fit(fit,cont.wt)
fit3<-eBayes(fit2)

DE<-topTable(fit3, coef=2 )

After this, The output is as follows:

 Gene_ID        logFC       AveExpr        t              P.Value            adj.P.Val        B
ACTB|60       12.59366   12.54151     202.8138  0.000000e+00  0.000000e+00 806.7855
EEF1A1|1915   12.06986 12.51399 187.5779  0.000000e+00  0.000000e+00 781.7838
ACTG1|71      11.93940 12.03115 179.5847  0.000000e+00  0.000000e+00 767.7521
UBC|7316      10.71139 11.15274 176.8877  0.000000e+00  0.000000e+00 761.7751
TPT1|7178     10.99882 11.58788 159.5321  0.000000e+00  0.000000e+00 728.9007
HSP90AB1|3326 11.00446 11.12925 157.1734 9.881313e-323 3.381237e-319 724.0502
FTH1|2495     10.98239 11.26717 153.0514 8.557019e-319 2.509774e-315 715.3888
EEF2|1938     10.82150 11.46502 151.3403 3.886332e-317 9.973786e-314 711.5412
PSAP|5660     10.71044 11.06326 147.8964 9.572234e-314 2.183639e-310 703.8942
HSP90AA1|3320 10.74747 10.94257 144.5401 2.294330e-310 4.710489e-307 696.4700

My Question: I am getting only a list of 10 genes, I am not able to pull all list. And, I want someone to validate my codes and method followed. Let me remind you all, I am a novice in coding/Bioinformatics. Please let me know if I am coding it correct or should I modify it.

Thanks a lot for your help.

-Ateeq Khaliq