[BioC] Inquiries on recommended design formulae for various experiments

[Yoong [guest]]

Hi there, 

I am currently making multiple comparisons using contrast in DESeq2. I am interested in differential expression for genes underlying germination mechanism due to high temperature. Here's my experimental design information: 

Genotypes: 4 different genotypes
Timepoint: 3 different timepoints 
Temperature: Low and high temperatures
3 biological replicates for each condition. 

I have a few questions regarding contrast function in DESeq2 package. My questions are mainly based on the table (Recommended design formulae for various experiments) in your package (Dec 23rd, 2013, page 11). I understand the terms 'condition', 'factor level', and 'group' are being used vaguely for flexibility purpose. I just want to make sure I am interpreting the terms correctly based on my experimental design. Here are my questions: 

1. >=3 level factor ’condition’: compare levels against another ~condition, or ~group + condition.
Am I correct to assume that I will be comparing different timepoints for ONE genotype. For example, timepoints: 6hours, 12hours, and 24hours after imbibition for Genotype A?  Alternatively, I can also compare ONE timepoint for four different genotypes. Am I right?

2. >=3 level factor ’condition’: compare significance of all levels ~condition, or ~group + condition.
My interpretation is the same as above (#1). But, instead of comparing gene counts, I will be comparing p=adjusted values?

3. 2 level factor ’condition’ but ’group’ has >= 3 levels.
Is it correct to assume that 'group'= genotypes (Genotype A, B, C, and D). The level factor 'condition' is Low and High temperatures. So, for this comparison, I will be comparing all four different genotypes for two different levels of temperatures (Low versus High). Am I correct?

4. Interactions between ’group’ and ’treatment’ ~group + treatment + group:treatment.
For this, just as an example, I will be comparing Genotype A at timepoint #1 with genotype B at timepoint #2? 

5. Time series: changes due to treatment after time 0.
For time series, I will be comparing changes in Genotype A at timepoints #1,#2, and #3 due to High temperature? Am I correct?

I apologize for my long questions. Thank you so much for your time and input!

Regards,
Yoong

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