How to deal with elimination of genes when using filterByExpr function?
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anikng • 0
@anikng-22672
Last seen 8 months ago

If I look the raw countdata, ~55% of my genes have zero counts in all the samples (64 samples in total for 8 genotypes). Though filterByExpr effectively eliminated several high FC yielding but low level expressed unwanted genes, I noticed that some of the biologically interesting genes are eliminated. For ex, if I take a subset of the samples for a single genotype having two time points for control and stress, following pattern (ie, 2h stress induction) is interesting to my biological question,

             2h_C1_1   2h_C1_2   4h_C1_1   4h_C1_2   2h_S1_1   2h_S1_2   4h_S1_1   4h_S1_2
Gene_x           0        0      18          15        48         54       29       35

How can I deal with elimination?

I use code,

group <- factor(paste(targets$Treat,targets$Time,targets$Genotype,sep="."))
cbind(targets,Group=group)
y <- DGEList(counts=x)

keep <- filterByExpr(y)
y <- y[keep, , keep.lib.sizes=FALSE]
y <- calcNormFactors(y)

design <- model.matrix(~0+group)
colnames(design) <- levels(group)

y <- estimateDisp(y, design)
fit <- glmQLFit(y, design)

my.contrasts <- makeContrasts(--contrast--,levels=design)
qlf <- glmQLFTest(fit, contrast=my.contrasts[--contrast--])
topTags(qlf)
edger DEGs normalization • 240 views
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3
Entering edit mode
@gordon-smyth
Last seen 3 hours ago
WEHI, Melbourne, Australia

To use filterByExpr correctly, you have to give it the design matrix or group as an argument:

keep <- filterByExpr(y, group=group)

Or alternatively, filterByExpr will detect the group automatically if you set:

y <- DGEList(counts=x, group=group)
keep <- filterByExp(y)

If you don't specify the groups at all, then filterByExpr will think that all samples are one group and will end up doing too much filtering.

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Entering edit mode

Now the list looks perfect..Thank you Gordon Symth!

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