I am working on RNA-Seq data which consists of 10 samples. My model has two factor: Time and Genotype, having 3 time point: Tbase, T45, Tend and 2 genotypes: WT, mutant (for Tend and Tbase each genotype has 2 replicates, for T45 each genotype has a single replicate).
I used the LRT test of DESeq2 v.1.6.3 since I am interested in the ratio of ratios results:
- (mutant_Tend /mutant_Tbase) / (WT_Tend /WT_Tbase)
- (mutant_T45 /mutant_Tbase) / (WT_T45 /WT_Tbase)
The commands that I used are:
> dds = DESeqDataSetFromMatrix(countData, colData, design = ~ Time + Genotype + Genotype:Time)
> dds = DESeq( dds, test = "LRT", reduced = ~ Time + Genotype)
 "Intercept" "Time_T45_vs_Tbase" "Time_Tend_vs_Tbase" "Genotype_mutant_vs_WT" "TimeT45.Genotypemutant"
> res_Tend_vs_Tbase = results(dds_LRT, name = "TimeTend.Genotypemutant")
> res_T45_vs_Tbase = results(dds_LRT, name = "TimeT45.Genotypemutant")
I have 3 questions:
1. Is this the correct way to receive the comparisons that I am interested in?
2. If so, I am also interested in comparing the two time points: Tend vs T45, what is the correct way to call results function for that comparison?
3. For both results: res_Tend_vs_Tbase, res_T45_vs_Tbase, I received different log2FoldChange values but the pvalue and padj columns are identical in both comparisons. Is there an explanation to that?
In case the sessionInfo() output is neede:
R version 3.1.1 (2014-07-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
 LC_COLLATE=Hebrew_Israel.1255 LC_CTYPE=Hebrew_Israel.1255 LC_MONETARY=Hebrew_Israel.1255 LC_NUMERIC=C
attached base packages:
 parallel stats4 stats graphics grDevices utils datasets methods base
other attached packages:
 DESeq2_1.6.3 RcppArmadillo_0.4.650.1.1 Rcpp_0.11.4 GenomicRanges_1.18.4 GenomeInfoDb_1.2.4
 IRanges_2.0.1 S4Vectors_0.4.0 BiocGenerics_0.12.1
loaded via a namespace (and not attached):
 acepack_1.3-3.3 annotate_1.44.0 AnnotationDbi_1.28.1 base64enc_0.1-2 BatchJobs_1.5 BBmisc_1.9
 Biobase_2.26.0 BiocParallel_1.0.3 brew_1.0-6 checkmate_1.5.1 cluster_2.0.1 codetools_0.2-10
 colorspace_1.2-4 DBI_0.3.1 digest_0.6.8 fail_1.2 foreach_1.4.2 foreign_0.8-63
 Formula_1.2-0 genefilter_1.48.1 geneplotter_1.44.0 ggplot2_1.0.0 grid_3.1.1 gtable_0.1.2
 Hmisc_3.15-0 iterators_1.0.7 lattice_0.20-30 latticeExtra_0.6-26 locfit_1.5-9.1 MASS_7.3-39
 munsell_0.4.2 nnet_7.3-9 plyr_1.8.1 proto_0.3-10 RColorBrewer_1.1-2 reshape2_1.4.1
 rpart_4.1-9 RSQLite_1.0.0 scales_0.2.4 sendmailR_1.2-1 splines_3.1.1 stringr_0.6.2
 survival_2.38-1 tools_3.1.1 XML_3.98-1.1 xtable_1.7-4 XVector_0.6.0
Thank you very much for the help,