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Question: Allele-specific expression with different gene lenghts
0
gravatar for gtechbio
2 days ago by
gtechbio0
gtechbio0 wrote:

Hi,
I want to use DESeq2 for assessing allele-specific expression. I will compare gene-level allelic counts of a yeast hybrid (basically comparing counts of orthologous genes of parentals of this hybrid).
I'm aware of this tutorial http://rstudio-pubs-static.s3.amazonaws.com/275642_e9d578fe1f7a404aad0553f52236c0a4.html.

My question is that parentals might have orthologous genes with different lengths, so for example ortholog in parent A is 1000bp and in parent B is 1020 bp. Is it possible at some step of DESeq2 to account for this length difference?

Thanks

ADD COMMENTlink modified 1 day ago by Michael Love17k • written 2 days ago by gtechbio0
1
gravatar for Michael Love
1 day ago by
Michael Love17k
United States
Michael Love17k wrote:

If you provide a matrix at the beginning of the analysis called “avgTxLength”, the analysis will control for gene length changes across sample. This should happen before running DESeq().

assays(dds)[["avgTxLength"]] <- lengths
ADD COMMENTlink written 1 day ago by Michael Love17k

Hi Michael,

Thanks for reply.

Just to make sure I do everything in order:

My `count_table` looks like

                        SC1    SC2    SC3    SU1    SU2    SU3
YBR177C_Sbay_4.429      2711   3397   3765   1246   2156   3287
YIL140W_Sbay_9.48         178    111    204    109    271    250
YLR268W_Sbay_10.366    1208   1170   1704    631    825    865

The code is

groups<-factor(x=c(rep("SC",3), rep("SU",3)), levels=c("SC","SU"))

colData <- DataFrame(condition=groups)

dds <- DESeqDataSetFromMatrix(count_table, colData, formula(~condition))
assays(dds)[["avgTxLength"]] <- as.matrix(length_data)   ### correcting for gene length

`length_data` looks like

                       SC1   SC2   SC3   SU1   SU2   SU3
YBR177C_Sbay_4.429     1353  1353  1353  1359  1359  1359
YIL140W_Sbay_9.48      2469  2469  2469  2457  2457  2457
YLR268W_Sbay_10.366     642   642   642   642   642   642
sizeFactors(dds) <- c(as.numeric(rep("1",6)))   ### this is allelic expression, so everything comes from the same library
dds <- DESeq(dds)
res <- results(dds)

Is everything correct?
Cheers and sorry if formatting is a bit messed

EDIT: When I omit sizeFactors(dds) <- c(as.numeric(rep("1",6))), I get the message `using 'avgTxLength' from assays(dds), correcting for library size`, but if I do both preset library size to 1 and feed the code with gene length, I do not receive the message above. Does it mean that with predefined size factors its not possible to account for gene length?

ADD REPLYlink modified 12 minutes ago • written 36 minutes ago by gtechbio0
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