Question: Differential Methylation Analysis using continuous outcome
0
daniellemo0 wrote:

Hi there,

I am trying to do a differential methylation analysis of probes in R using "days to death" as my clinical factor. I have values ranging from 9 to 3258 days and need to make contrasts between these values, but there are too many values to do this manually. Is there a automatic way to contrast a wide range of continuous values? If not, can I specify ranges (i.e. 0-500, 501-1000 days, etc.) and input this into the "makecontrasts" function (not sure how to go about doing this). Below is the code I am using.

``````outcome <- factor(targets\$days_to_death)
design <- model.matrix(~0+outcome, data=targets)
colnames(design) <- c(levels(outcome))
fit <- lmFit(MVals_d2d, design)
contMatrix <- makeContrasts(**?????**,
levels=design)
fit2 <- contrasts.fit(fit, contMatrix)
fit2 <- eBayes(fit2)
summary(decideTests(fit2))
ann450kSub <- ann450k[match(rownames(mVals),ann450k\$Name),
c(1:4,12:19,24:ncol(ann450k))]
DMPs <- topTable(fit2, coef=NULL, adjust.method="fdr", p.value=0.05, genelist=ann450kSub)
``````

modified 11 months ago by Charles Warden7.5k • written 11 months ago by daniellemo0
2
Kevin Blighe52k wrote:

You could encode `targets\$days_to_death` as a categorical variable with `500` (0-500), `1000` (501-1000), `1500` (1001-1500), et cetera, and then your contrasts would be, for example:

``````makeContrasts(1000-500, levels=design)
makeContrasts(1500-500, levels=design)
*et cetera*
``````

If you need help to categorise your `targets\$days_to_death`, then use `symnum()` (stats):

``````input <- c(0,1,2,3,4,5,6,7,8,9,10)
cuts <- c(0, 2.5, 5, 7.5, 10)
encoding <- c('<=2.5', '<=5', '<=7.5', '<=10')

c(stats::symnum(input,
corr = FALSE,
na = FALSE,
cutpoints = cuts,
symbols = encoding))

 <=2.5 <=2.5 <=2.5 <=5   <=5   <=5   <=7.5 <=7.5 <=10  <=10  <=10
``````

Kevin

1
Charles Warden7.5k wrote:

It looks like you are fairly familiar with R, which is great!

If not, here are a couple pointers:

1) You can do continuous variable analysis in COHCAP (with the `COHCAP.site()` and `COHCAP.avg.by.island()` functions) by setting `ref="continuous"`

The delta-beta (or delta percent methylation) threshold is a little tricker to define in this situation, but you can play around with the `lower.cont.quantile` and `upper.cont.quantile` values.

I think I've only tested it with one or two projects, but setting `alt.pvalue = "RcppArmadillo.fastLmPure"` should noticeably speed up the analysis.

2) Even if you don't use COHCAP directly, you can use the COHCAP code to see how this analysis is performed.

For example, the code is fairly long for the `COHCAP.site()` function, but the smaller functions used various analyses are towards the top: https://github.com/cwarden45/COHCAP/blob/master/R/COHCAP.site.R