Setting Contrast for DESeq2 results
I am trying to recreate this heatmap. How would I compare the four variables: Ly49+/- and MOG/MOGSP to get a single heatmap? Thank you for helping me through this.
I need to compare both Ly49+ versus Ly49- and MOG versus MOG plus SP. Column data in at the bottom. The function I used for DESeq2 is,
dds = DESeqDataSetFromMatrix(countData=countData,
colData=colData,
design= ~ cell_treatment)
I am not sure if the design is right. For the results, how should I write the contrasts? I am very confused and would really appreciate any help!
DataFrame with 11 rows and 2 columns
cell treatment
<character> <character>
MOGSP_2 SPtetramer+CD8+TCell MOGSP
MOGSP_3 SPtetramer+CD8+TCell MOGSP
MOG_3 SPtetramer+CD8+TCell MOG
MOG_4 SPtetramer+CD8+TCell MOG
MOG_5 SPtetramer+CD8+TCell MOG
Ly49N_1 Ly49-CD8+TCell MOGSP
Ly49P_1 Ly49+CD8+TCell MOGSP
Ly49N_2 Ly49-CD8+TCell MOGSP
Ly49P_2 Ly49+CD8+TCell MOGSP
Ly49N_3 Ly49-CD8+TCell MOGSP
Ly49P_3 Ly49+CD8+TCell MOGSP
Thank you!
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mahejabeen.nidhi •
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For DESeq2 you need to define two groups for doing DE analysis. It seems you have more than two groups for comparison. My suggestion is to keep out everything out from your analysis (edit: differential expression analysis) except the first two groups that you want to know genes within the two. Then you may go to compare the next two groups.
I am trying to recreate this heatmap https://www.nature.com/articles/s41586-019-1467-x/figures/6 How would I compare the four variables: Ly49+/- and MOG/MOGSP to get a single heatmap? Thank you for helping me through this.
General steps for creating an expression heatmap is :
1-converting your normalized expression matrix to z score, 2- clustering and plotting your data. You may find this tutorial from Kevin Blighe helpful. Indeed you can find an answer for most of your questions in this field by following Kevin's activities on Biostars.
Subsetting your dataset down to the two groups you want to compare is not necessary, or suggested. It's preferred to keep all the samples together for better library normalization and dispersion estimation, and the specify which subsets to compare to each other with the contrasts.
Added an edit to be more accurate.
Can you clarify? So these Ly49(+/-) cells all received that MOGSP treatment, right? And the other five samples have 2 with MOGSP and 3 without it? Is this the same batch of experiments? Currently it seems that MOG is nested with the status of
SPtetramer+CD8+TCellso do you want to compare the three MOG with all 8 MOGSPs? I guess for the Ly49 you want to compare only within the bottom 6? Please clarify about the experimental setup.Hello mahejabeen.nidhi!
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