RNA Seq normalization

[luoye]

When you first use DESeq, you combine a table of counts and a list of conditions to create a count data set

cds <- newCountDataSet(countTable,conditions)

You can give the count data set your own size factors using

sizeFactors(cds) <- #input

If you wanted to use TMM normalized sizeFactors from EdgeR rather than those given by DESeq then you can first:

x <- calcNormFactors(as.matrix(countTable)

and then give this to the count data set:

sizeFactors(cds) <- x

thank you very much,i do as you say,but the result is not what i expect.

[chadn737]

sorry,i can not understand what you mean,can you tell me some more detail？
did you mean is: cds=calcNormFactors(cds) ,sizeFactors(cds)?
thank you very much.

When you first use DESeq, you combine a table of counts and a list of conditions to create a count data set

cds <- newCountDataSet(countTable,conditions)

You can give the count data set your own size factors using

sizeFactors(cds) <- #input

If you wanted to use TMM normalized sizeFactors from EdgeR rather than those given by DESeq then you can first:

x <- calcNormFactors(as.matrix(countTable)

and then give this to the count data set:

sizeFactors(cds) <- x

[luoye]

Do the same thing with the normalization factors from EdgeR. You can even feed DESeq the normalization factors from EdgeR by using sizeFactors(cds)= normalization factors from EdgeR

sorry,i can not understand what you mean,can you tell me some more detail？
did you mean is: cds=calcNormFactors(cds) ,sizeFactors(cds)?
thank you very much.

[chadn737]

Do the same thing with the normalization factors from EdgeR. You can even feed DESeq the normalization factors from EdgeR by using sizeFactors(cds)= normalization factors from EdgeR

[luoye]

You can use EdgeR to get TMM normalized data using calcNormFactors() in R.

What do you want to use the normalized data as input for?

hi chadn737
thank you very much for your reply,I mean is that when i use EdgeR to get TMM calcNormFactors() in R to nomalization ,i want to see the difference
between normalized data and the raw data .for example ,In DESeq, you get normalized counts by dividing the raw counts by the appropriate size factor.but in edgeR ,how can i do this normalized counts ?
thank you

[chadn737]

hi,everyone
i want to use TMM method to normalization,but i encounter a question ,how can i get the normalized counts after TMM ,thank you very much.

You can use EdgeR to get TMM normalized data using calcNormFactors() in R.

What do you want to use the normalized data as input for?

[luoye]

hi BENM,
i meant the later one.
thank you for providing the link to this old post. that what i was looking for.thanks.

hi,everyone
i want to use TMM method to normalization,but i encounter a question ,how can i get the normalized counts after TMM ,thank you very much.

[harshinamdar]

hi BENM,
i meant the later one.
thank you for providing the link to this old post. that what i was looking for.thanks.

[BENM]

hi, do you mean experiment normalization or data normalization for quantification analysis?

If it is for cDNA libraries normalization, one of application is duplex-specific nuclease (DSN), which is based on the kinetics of cDNA reassociation. (refers to: P. A. Zhulidov, etc. al., A Method for the Preparation of Normalized cDNA Libraries Enriched with Full-Length Sequences. Russian Journal of Bioorganic Chemistry, Vol. 31, No. 2, 2005. and Irina Shagina, etc. al., Normalization of genomic DNA using duplex-specific nuclease. BioTechniques 48:455-459, June 2010)

Or the later, there is two general formulas for RNA-seq data normalization: RPKM (reads per kilobase per millions of reads mapped) and FPKM (fragments per kilobase per million mapped fragments), and an useful tool - Cufflinks. You can follow the previous post in SEQanswer to find more details: RNA-seq and normalization numbers (http://seqanswers.com/forums/showthr...p?t=586&page=1)

[harshinamdar]

what do you understand by normalization of RNA seq data? what are the tools available for it?