Dear Bioconductor community,
I have timecourse RNA-seq data.
Animals received 4 differents treatments (group = GpA, GpB, GpCD, GpE).
There are 6 animals in each group.
For each animal,blood were collacted before any treatment (D0) and at 3 time points after treatment: D3, D7 and D28.
So, i have a design matrix looking like :
Animal Time Treatment Rep M657 0 GpA 1 M657 3 GpA 1 M657 7 GpA 1 M657 28 GpA 1 M658 0 GpA 2 M658 3 GpA 2 M658 7 GpA 2 M658 28 GpA 2 . . . . D627 0 GpB 1 D627 3 GpB 1 D627 7 GpB 1 D627 28 GpB 1 D628 0 GpB 2 D628 3 GpB 2 D628 7 GpB 2 D628 28 GpB 2 . . . .
With rep being a factor value for Animal-Group which distinguishes the individual nested within a group.
I want to determine :
1/ Which genes respond at either the Day3, Day7 or day28 in the different group
for exemple :
GpA : Day3vsDay0 Day7vsDay0, Day28vsDay0
do I have to analyse each treatment separatly ? Or I can analyse them all together ?
Because each subject yields a value for each time point, can I take "Animal" effect into my model ?
2/ Which genes respond differently over time in the different groups ie :
Which genes respond differently over time in the GpA relative to the GpB?
Here is what I did :
Design_clean <- data.frame(Treatment=Group,Time=factor(Time), ind.n=factor(order$Rep),row.names=row.names(order)) dds <- DESeqDataSetFromMatrix(countData = Count, colData = Design_clean, design=~Treatment+Treatment:ind.n+Treatment:Time) dds <- DESeq(dds, minReplicatesForReplace=Inf, modelMatrixType="standard", parallel=TRUE)
Is the design correct ?
Here are a subset of resultsNames(dds) output :
If I understant well, the following comparisons answer to my first question ie, which genes respond at either the Day3, Day7 or Day28 in the different group :
"GpA.Time3" "GpA.Time7" "GpA.Time28"
It's not indicated if the comparison was made against baseline (ie Day0), is it ? Is the within subject variability taken into account in these comparison ?
I also have :
"GpA.ind.n1" "GpA.ind.n2" "GpA.ind.n3" "GpA.ind.n4" "GpA.ind.n5" "GpA.ind.n6"
Is it within subject variability ?
"GpA_vs_GpB" "GpA_vs_GpCD" "GpA_vs_GpE"
Here, the comparisons are made between the Group, but Time information is not take into account.
How can I do, to have, for example, genes that respond differently over time in the GpA relative to the GpB at day 3 ie GpA_vs_GpB.day3 = (GpA.3-GpA.0)-(GpB.3-GpB.0)
Thanks in advance for your help,