Question

Design formula for pairwise comparison of experimental setup with multiple factors

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2021yearsold • 0

@94673e37

Last seen 3.9 years ago

I have RNAseq with multiple factors such as 4 genotypes, 2 treatment and 4 time points. The top few lines look like,

 Samples    Genotype    Treatment   Time
 S1        VN1            Control   1hr
 S2        VN1            Control   1hr
 S3        VN1            Stress    1hr
 S4        VN1            Stress    1hr
 S5        VN1            Control   3hr
 S6        VN1            Control   3hr
 S7        VN1            Stress    3hr
 S8        VN1            Stress    3hr
 .
 .

Now I am estimating DEGs using DESeq2 package. My intention is to make pairwise comparisons to see time effect and treatment effect using different combinations. For example,

VN1 at 1hr in Control Vs VN1 at 1hr in Stress
VN1 at 1hr in Control Vs VN1 at 3hr in Control

Since the experimental setup includes multiple factor,I am a bit confused about the correct design for my purpose. Can anyone help?

DEGs DESeq2 • 1.2k views

ADD COMMENT • link 4.0 years ago 2021yearsold • 0

score 2 · Accepted Answer · 2021-01-05

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Entering edit mode

swbarnes2 ★ 1.4k

@swbarnes2-14086

Last seen 2 days ago

San Diego

To compare small subgroups of your data, do what the vignette says here. (even though the section is on interactions, your questions do not involve using interactions)

http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions

You need to make a new column of sample data that combines the others, then make that your design, and contrast one subgroup with another.

ADD COMMENT • link 4.0 years ago swbarnes2 ★ 1.4k

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Thanks. I combined all three factors into one and used contrast function to make comparisons among subgroups. Is the following method correct?

dds <- DESeq(dds)
res1 <-results(dds, contrast=c("condition","VN1.C.3h","VN1.C.1h"), independentFiltering=TRUE, alpha=0.05, pAdjustMethod="BH", parallel=TRUE)
res1 <- lfcShrink(dds, contrast=c("condition","VN1.C.3h","VN1.C.1h"), res=res1)
write.csv(as.data.frame(res1), file="VN1.C.3hVsVN1.C.1h.csv")

ADD REPLY • link 4.0 years ago 2021yearsold • 0