I seek help from forum members in identifying the most appropriate analysis tools / software for performing the following analyses on the following time-series NGS RNA-Seq count data (total of 144 libraries):
- 9 time points,
- 4 genotypes, and
- 4 replicates each.
I've filled in answers to some of my questions, but please do add / comment if you think any of those answers should be different.
Q1. Differential Gene Expression (DGE) across entire time series, between genotypes
Q2. DGE at a specific time point, between genotypes
Just DESeq2 (pairwise) ?
Q3. DGE between different time points, within same genotype
Again, just DESeq2 (pairwise comparison) ?
Q4. Reporting Gene Co-Expression Networks (GCNs) for time-series data from one genotype (let's call this baseline)
Q5. Reporting which GCNs are broken / altered in 'test' genotype vs. 'baseline' genotype
Q6. Reporting temporal shifts in expression of GCN components in test genotype vs. baseline data
Q7. Reporting quantitative shifts in expression of GCN components in test genotype vs. baseline data
Q8. Some genes are going to be cyclic and they could confound inferences, right? How is this controlled?
Q9. And some other genes will be shifted in expression strength and/or timing, and these could also confound analyses? Again, how is this problem solved?
Open ended question: Furthermore, what are other considerations to keep in mind while performing time-series analyses and for choosing the correct tools for answering questions 1 - 7 listed above?
I am starting to practice using DESeq2 and WGCNA on my read counts, but I am not entirely sure these are the best choices to answer my questions. I look forward to all your replies. Thanks again!