Question

Removing Unwanted Variations

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AZ ▴ 30

@fereshteh-15803

Last seen 18 months ago

United Kingdom

Hi

I have treatment versus control collected in eight batches (NanoString transcriptome)

I have tried NanoStringNCTools and RUVseq to remove unwanted variations

Here I suppose my eight batches can introduce unwanted technical variations

I have twenty housekeeping genes in my panel using them I want to combat with unwanted variations

So,

## ----ruv_spikes, fig.cap="RUVg normalization based on spike-in controls.", fig.subcap=c("RLE plot","PCA plot")----
set1 <- RUVg(set, spikes, k=1)
pData(set1)

enter image description here

> set1$x
 [1] 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 4
[29] 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 7 7
[57] 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8
Levels: 1 2 3 4 5 6 7 8

Please ,if you just tell me what W_1 is embedded in set1?

I know x is the main design ; But I am sure W_1 comes from where?

Is that correcting factor based on spikes in gene?

For instance as a part of tutorial you mention

This is done by considering a design matrix that includes both the covariates of interest (here, the treatment status) and the factors of unwanted variation.

design <- model.matrix(~x + W_1, data=pData(set1))

I have both covariate of interest and some some batches, so shall I still include the batch in the formula?

What would be a formula comparing treatment/control, removing unwanted variation and deducing batches?

I am desperately confused

Thanks for any clarification

DESeq2 NanoStringNCTools RUVnormalize RUVSeq DifferentialExpression • 1.1k views

ADD COMMENT • link 19 months ago AZ ▴ 30

score 1 · Answer 1 · 2023-04-12

1

Entering edit mode

ATpoint ★ 4.5k

@atpoint-13662

Last seen 9 hours ago

Germany

If you precisely know the batches, have you checked whether a) there is in fact evidence for batch effects in a PCA (see vignette) and b) if so then to simply include the batch information as a factor into the design rather than using more complicated methods like RUVseq? For a) I would normalize first to the controls using the controlGenes option in estimateSizeFactors(), then run vst() and then plotPCA() (or any PCA approach), coloring points by known batch. I would only use RUVseq (which has the downside that the choice of k is arbitrary) if the known batches cannot explain the unwanted variation.

ADD COMMENT • link 19 months ago ATpoint ★ 4.5k

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Entering edit mode

Thanks a million

I have done so

> dds <- DESeqDataSetFromMatrix(countData = counts(set),
+                               colData = pData(set),
+                               design = ~ Status_2) #counts(set)[1:652,], ##use design with W_1 or W_2 based on k value

> dds <- estimateSizeFactors(dds, controlGenes = rownames(dds) %in%rownames(housekeeping))
> dds <- DESeq(dds)
using pre-existing size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
-- replacing outliers and refitting for 20 genes
-- DESeq argument 'minReplicatesForReplace' = 7 
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing
> vsd <- varianceStabilizingTransformation(dds)
> dds
class: DESeqDataSet 
dim: 784 73 
metadata(1): version
assays(6): counts mu ... replaceCounts replaceCooks
rownames(784): CCNO MYC ... G6PD ERCC3
rowData names(23): baseMean baseVar ... maxCooks replace
colnames(73): 20210202_annels-3-020221_01_04.RCC 20210202_annels-3-020221_01_05.RCC ...
  20221014_cls-1-141022_01_10.RCC 20221014_cls-1-141022_01_11.RCC
colData names(15): Bacth_no Samp_no ... sizeFactor replaceable
> plotPCA(vsd, intgroup=c("Status_2", "Bacth_no"))
> pcaData <- plotPCA(vsd, intgroup=c("Status_2", "Bacth_no"), returnData=TRUE)
> percentVar <- round(100 * attr(pcaData, "percentVar"))
> ggplot(pcaData, aes(PC1, PC2, color=Bacth_no, shape=Status_2)) +
+   geom_point(size=3) +
+   xlab(paste0("PC1: ",percentVar[1],"% variance")) +
+   ylab(paste0("PC2: ",percentVar[2],"% variance")) + 
+   coord_fixed()
> pcaData <- plotPCA(vsd, intgroup=c("Bacth_no"), returnData=TRUE)
> percentVar <- round(100 * attr(pcaData, "percentVar"))
> ggplot(pcaData, aes(PC1, PC2, color=Bacth_no)) +
+   geom_point(size=3) +
+   xlab(paste0("PC1: ",percentVar[1],"% variance")) +
+   ylab(paste0("PC2: ",percentVar[2],"% variance")) + 
+   coord_fixed()
>

I have plotted PCA for different things in my colData

The number of batches we collected the data before using housekeeping genes enter image description here