[BioC] Help With RNA-seq

[Valerie Obenchain]

Hi Tina,

It's difficult to help without knowing what your data look like or what 
error message you are seeing. Both pieces of information would be helpful.

For starters I think you need to provide 'replicate' and 'groups' 
arguments when you create your new "countData" object. Depending on what 
order your data are in you need something like,

     groups <- list(NDE = c(1,1,1,1,1,1,1,1,1,1), DE = 
c(1,2,1,2,2,1,2,1,2,1))
     replicates <- c("Kidney", "Liver", "Kidney", "Liver", "Liver', 
"Kidney", "Liver", "Kidney", "Liver", "Kidney")

Then create your "countData" with these variables,

     CD <- new("countData", data = as.matrix(MA.subsetA$M), libsizes = 
as.integer(d$samples$lib.size),
         replicates = replicates, groups = groups)

Now look at the CD object and make sure the columns are labeled as they 
should be and the other slot values make sense. The MA plot call would 
look something like,

     plotMA.CD(CD, samplesA = "Kidney", samplesB = "Liver")

The author used the red and black colors for the vignette plot because 
there was a known structure to the data; the first 100 counts showed 
differential expression and the last 900 did not. You probably have a 
different situation in your data so using the same color scheme may not 
make sense.

Valerie


On 01/23/2012 06:13 AM, Tina Asante Boahene wrote:
> Hi all,
>
> I am conducting some analysis using the Marioni et al data.
>
> However, I am having a bit of trouble using my data to conduct the analysis based on the baySeq package.
>
>   And I was wondering if you could stir me in the right direction.
>
> I have already used edgeR to find the library sizes for the ten libraries I have for my data as well as for the groups (Liver and Kidney) as stated below.
>
>
> library(baySeq)
> library(edgeR)
> library(limma)
> library(snow)
>
> cl<- makeCluster(4, "SOCK")
>
>
> ##Calculating normalization factors##
> D=MA.subsetA$M
> head(D)
> names(D)
> dim(D)
>
> g<- gsub("R[1-2]L[1-8]", "", colnames(D))
> d<- DGEList(counts = D, group = substr(colnames(D), 5, 30))
> d$samples
> names(d)
> dim(d)
>
>
> I will like to know how to model my code in order to produce the MA plot for count data
>
>
> This is what I have, however it runs with the wrong response.
>
> Can someone help me fix this please.
>
> CD<- new("countData", data = as.matrix(MA.subsetA$M), libsizes = as.integer(d$samples$lib.size))
>
> plotMA.CD(CD, samplesA = 1:5, samplesB = 6:10, col = c(rep("red",
> 100), rep("black", 900)))
>
>
> How can I get it to recognise the "groups" as "g" (Library and Kidney)
>
> This is the output for the groups   [1] "Kidney" "Liver"  "Kidney" "Liver"  "Liver"  "Kidney" "Liver"  "Kidney"  "Liver"  "Kidney"
>
> thank you.
>
>
>
>
>
>
> Kind Regards
>
> Tina
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