Need Help Doing Complex Contrasts in DESeq2

pthom010 · 05-21-2021, 10:43 AM

I recently completed a 22 day time course with a resistant and susceptible plant with five timepoints (0hr - control), 1dpi, 7dpi, 14dpi, and 22dpi. I was able to do three replicates and sequenced my smaples t get RNAseq libraries (30 libraries total). I now have begun my RNA seq analysis in DESeq2. I have set up my samples object for the comparisons like so:

Code:

sample

         timepoint treatment genotype
S1A_rep1         0        SA        S
S1A_rep2         0        SA        S
S1A_rep3         0        SA        S
S1B_rep1         1        SB        S
S1B_rep2         1        SB        S
S1B_rep3         1        SB        S
S1C_rep1         7        SC        S
S1C_rep2         7        SC        S
S1C_rep3         7        SC        S
S1D_rep1        14        SD        S
S1D_rep2        14        SD        S
S1D_rep3        14        SD        S
S1E_rep1        22        SE        S
S1E_rep2        22        SE        S
S1E_rep3        22        SE        S
R1A_rep1         0        RA        R
R1A_rep2         0        RA        R
R1A_rep3         0        RA        R
R1B_rep1         1        RB        R
R1B_rep2         1        RB        R
R1B_rep3         1        RB        R
R1C_rep1         7        RC        R
R1C_rep2         7        RC        R
R1C_rep3         7        RC        R
R1D_rep1        14        RD        R
R1D_rep2        14        RD        R
R1D_rep3        14        RD        R
R1E_rep1        22        RE        R
R1E_rep2        22        RE        R
R1E_rep3        22        RE        R

I am using my treatment column to design my comparisons. I have done the following comparisons so far:

RA-SA
RB-SB
RC-SC
RC-SD
RE-SE
RB-RA
RC-RA
RD-RA
RE-RA
SB-SA
SC-SA
SD-SA
SE-SA

My DESEq2 comparison is done withe the follwing code:

Code:

dds <- DESeqDataSetFromTximport(txi, sample, ~treatment)

And my results are retrieved like so:

Code:

results(deseqData, contrast=c("treatment","RB","RA"))

I now want to do the following comparisons:

Code:

(RB - SB) - (RA - SA) (compare RB and SB, then compare that to RA and SA, compared)
(RE + SE) - (RA + SA)
(RE + RD) - (RC + RB)
[(RE + RD) - (RC + RB)] - [(SE + SD) - (SC - SB)] (compare the early vs late response of the R plants to that of the susceptible)

I am not sure of what code to use. Would anybody be able to point me in the right direction? Any help appreciated.

BigOmics · 09-22-2021, 04:37 AM

You have probably solved your issue by now, but if you still struggle with your transcriptomic data, you could try using our Omics Playground platform. We have an interactive GUI that allows you to generate pairwise comparisons with a high degree of flexibility when you input your data into the platform. You can also intersect the results of pairwise comparisons and represent them as Venn diagrams and scatterplots.

If you want to give it a shot, you can try our free access demo version: https://public.bigomics.ch/app/omicsplayground_contrib

Hope that addresses your need!

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05-21-2021, 10:43 AM	#1
pthom010 Junior Member Location: us Join Date: May 2021 Posts: 1	Need Help Doing Complex Contrasts in DESeq2 I recently completed a 22 day time course with a resistant and susceptible plant with five timepoints (0hr - control), 1dpi, 7dpi, 14dpi, and 22dpi. I was able to do three replicates and sequenced my smaples t get RNAseq libraries (30 libraries total). I now have begun my RNA seq analysis in DESeq2. I have set up my samples object for the comparisons like so: Code: sample timepoint treatment genotype S1A_rep1 0 SA S S1A_rep2 0 SA S S1A_rep3 0 SA S S1B_rep1 1 SB S S1B_rep2 1 SB S S1B_rep3 1 SB S S1C_rep1 7 SC S S1C_rep2 7 SC S S1C_rep3 7 SC S S1D_rep1 14 SD S S1D_rep2 14 SD S S1D_rep3 14 SD S S1E_rep1 22 SE S S1E_rep2 22 SE S S1E_rep3 22 SE S R1A_rep1 0 RA R R1A_rep2 0 RA R R1A_rep3 0 RA R R1B_rep1 1 RB R R1B_rep2 1 RB R R1B_rep3 1 RB R R1C_rep1 7 RC R R1C_rep2 7 RC R R1C_rep3 7 RC R R1D_rep1 14 RD R R1D_rep2 14 RD R R1D_rep3 14 RD R R1E_rep1 22 RE R R1E_rep2 22 RE R R1E_rep3 22 RE R I am using my treatment column to design my comparisons. I have done the following comparisons so far: RA-SA RB-SB RC-SC RC-SD RE-SE RB-RA RC-RA RD-RA RE-RA SB-SA SC-SA SD-SA SE-SA My DESEq2 comparison is done withe the follwing code: Code: dds <- DESeqDataSetFromTximport(txi, sample, ~treatment) And my results are retrieved like so: Code: results(deseqData, contrast=c("treatment","RB","RA")) I now want to do the following comparisons: Code: (RB - SB) - (RA - SA) (compare RB and SB, then compare that to RA and SA, compared) (RE + SE) - (RA + SA) (RE + RD) - (RC + RB) [(RE + RD) - (RC + RB)] - [(SE + SD) - (SC - SB)] (compare the early vs late response of the R plants to that of the susceptible) I am not sure of what code to use. Would anybody be able to point me in the right direction? Any help appreciated.

09-22-2021, 04:37 AM	#2
BigOmics Junior Member Location: Switzerland Join Date: Jul 2021 Posts: 1	You have probably solved your issue by now, but if you still struggle with your transcriptomic data, you could try using our Omics Playground platform. We have an interactive GUI that allows you to generate pairwise comparisons with a high degree of flexibility when you input your data into the platform. You can also intersect the results of pairwise comparisons and represent them as Venn diagrams and scatterplots. If you want to give it a shot, you can try our free access demo version: https://public.bigomics.ch/app/omicsplayground_contrib Hope that addresses your need!