This is a test version of Biostars. For the public version, visit https://www.biostars.org.
RNA seq normalization(TMM)

i have htseq-count data now i want to normalize it using TMM for diffrential expression analysis .can anybody tell me how to do this Thanking in anticipation

rna-seq r gene
• 9,524 views • link
updated by h.mon h.mon • written by maryak • maryak

1 answer

The edgeR User Guide is an excellent read, and has the answer to this question, and probably the answer to some questions you will have in the near future.

• 1 views • link
written by h.mon h.mon

can you please guide me in the form of code because i am new with R

• 1 views • link
written by maryak • maryak 
library(edgeR)
#TMM Normalisation
lib.size <- sum(d$sample1)
scale.factors <- calcNormFactors(TMM$sample1, method = "TMM")
norm.data <- t(t(TMM$sample1)/(scale.factors*lib.size))
write.csv(norm.data,"/home/TMM.csv")
• 0 views • link
written by k.kathirvel93 k.kathirvel93

lib.size <- sum(d$sample1) is it calculating library size of just one sample??? d is the name of file object that contain counts??

• 1 views • link
written by maryak • maryak

Read the edgeR User Guide, it has plenty of code examples to guide you through the whole analysis, from the very beginning to the very end. Then, if /when you have difficulties, show us how is your data (ideally, with a small example), tell us what you did (the code), and the error you got.

The Similar Posts links to the right are also a great resource, and indeed I found one link with code and explanations about TMM normalization which would probably be very helpful to you.

• 1 views • link
written by h.mon h.mon

i have run this code but no file is created even after the program is executed successfully and no error message appears 

library(edgeR)
seqdata <- read.delim("C:\\Users\\hp folio\\Desktop\\TCGA-DLBC.htseq_counts.tsv")
scale.factors <- calcNormFactors(seqdata,lib.size=NULL method = "TMM")
norm.data <- t(t(seqdata)/(scale.factors*lib.size))
write.csv(norm.data,"C:\\Users\\hp folio\\Desktop\\TMM.csv")
• 1 views • link
updated by h.mon h.mon • written by maryak • maryak

I suspect that you have a problem on write.csv line. Check your path is correct or not.

• 1 views • link
written by k.kathirvel93 k.kathirvel93

if path is incorrect at least it must show some error... and i checked there is no problem in path

• 1 views • link
written by maryak • maryak

if path is incorrect at least it must show some error... and i checked there is no problem in path

• 1 views • link
written by maryak • maryak

Log in to answer this question.

More posts like this