Plotting PCA plot in DESeq2 on SVA corrected counts
1
0
Entering edit mode
3.5 years ago
Mozart ▴ 330

Hello everyone, I am trying to understand how to show a PCA plot on SVA-corrected counts by using DESeq2 (I know that expert guys tend to prefer some other tools but I need this for a presentation). Many posts out there (like this, this and this) but none of them seem to address exactly what I need. Specifically, looking at the following post and applying an rlog on it, PCA plot doesn't show up any difference from the uncorrected version. Any useful hints?

Thanks

sva deseq2 pca • 1.3k views
ADD COMMENT
2
Entering edit mode
3.5 years ago
ATpoint 78k

I prefer to perform PCA outside of DESeq2 (but inspired by the code): Assume we start from a count matrix counts that should be on log scale. That could be your matrix of batch-corrected counts. I did not read the entire question because it was too extensive so tell me if I got you wrong.

# Find most variable genes, say top 500:
rv <- rowVars(as.matrix(counts))
select <- order(rv, decreasing = TRUE)[seq_len(min(500, length(rv)))]

# perform PCA on these genes:
pca <- prcomp(t(counts[select, ]))

# calculate the explained variation per principal component:
percentVar <- pca$sdev^2/sum(pca$sdev^2)

# feed into ggplot:
d <- data.frame(PC1 = pca$x[, 1], PC2 = pca$x[, 2], stringsAsFactors = FALSE)

# Most simplistic plot:
ggplot(data=d, aes(x = PC1, y = PC2)) + geom_point()

You can add legends etc. to indicate which data point is which sample etc.

ADD COMMENT
0
Entering edit mode

Thanks ATpoint, that basically solved my problem. It really sounds so reassuring how important this forum could be in the learning process. It's good to have a good string of code to use for all the pipelines, to be honest.

Thanks a million.

ADD REPLY
0
Entering edit mode

Sure think. Most of what I know today I learned from online platforms such as this one here, blogs as well as from the many Bioconductor manuals and packages.

ADD REPLY
0
Entering edit mode

Thanks for the useful hints ATpoint

Not sure why applying this further normalisation step before plotting results? Any ideas/recommendations? Difficult to catch up with her during this period of time, unfortunately.

ADD REPLY
0
Entering edit mode

I personally cannot comment since I am not an active user of this package, therefore no experience with it.

ADD REPLY

Login before adding your answer.

Traffic: 2479 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6