[Bioc-devel] DESeq2 time series - How to set the experimental design

[Michael Love]

hi Nadia,

The Bioconductor support site for users now lives here:
https://support.bioconductor.org

I've answered a couple of questions already on how to analyze time
series datasets with DESeq2, so maybe take a look around there and
feel free to post a question tagged with 'deseq2' if you still have
one.

Mike

On Wed, Oct 8, 2014 at 12:16 PM, Nadia Kamal
<nkamal at cebitec.uni-bielefeld.de> wrote:
>
> Hi,
>
> I am trying to analyze RNA-Seq Data with DESeq2 and could use some help. I have 2 genotypes and 14 timepoints. I want to find differences in gene expression between both genotypes overall and at every timepoint and between every two timepoints in each genotype.
>
> Here is what I did so far.
>
> library("DESeq2")
>
> directory<-"............."
> sampleFiles <- grep("??",list.files(directory),value=TRUE)
>
> time <- factor(c("T12", "T13", "T14", "T1", "T11", "T5", "T7", "T2", "T9", "T3", "T6", "T8", "T4", "T10", "T12", "T13", "T14", "T1", "T11", "T5", "T7", "T2", "T9", "T3", "T6", "T8", "T4", "T10" ))
> genotype <- factor(c("GF","GF","GF","GF","GF","GF","GF","GF","GF","GF","GF","GF","GF","GF","VB","VB","VB","VB","VB","VB","VB","VB","VB","VB","VB","VB","VB","VB"))
> sampleTable<-data.frame(sampleName=sampleFiles, fileName=sampleFiles,  genotype=genotype, time=time)
> ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable=sampleTable, directory=directory, design=~genotype+time+time:genotype)
>
> ddsHTSeq$genotype<-factor(ddsHTSeq$genotype, levels=c("GF","VB"))
> ddsHTSeq$time <- factor(ddsHTSeq$time, levels=c("T1", "T2", "T3", "T4", "T5", "T6", "T7", "T8", "T9", "T10", "T11", "T12", "T13", "T14"))
>
> dds <- DESeq(ddsHTSeq)
> res <- results(dds)
>
> ddsHTSeq <- estimateSizeFactors(ddsHTSeq)
> ddsHTSeq <- estimateDispersions(ddsHTSeq)
> ddsLRT <- nbinomLRT(dds, reduced = formula(~time+genotype))
>
> or ~genotype + time in the full and ~time in the reduced formula, but I think this is not suitable for my experiment. When I use the first design and do plotMA, I have no significant genes at all, so I must be doing something wrong. I would be greatfull for some help. Thank you.
>
> > sessionInfo()
> R version 3.1.1 (2014-07-10)
> Platform: x86_64-redhat-linux-gnu (64-bit)
>
> locale:
>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
>  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
>  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8
>  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
>  [9] LC_ADDRESS=C               LC_TELEPHONE=C
> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
>
> attached base packages:
> [1] parallel  stats     graphics  grDevices utils     datasets methods
> [8] base
>
> other attached packages:
> [1] DESeq2_1.4.5              RcppArmadillo_0.4.450.1.0
> [3] Rcpp_0.11.2               GenomicRanges_1.16.4
> [5] GenomeInfoDb_1.0.2        IRanges_1.22.10
> [7] BiocGenerics_0.10.0
>
> loaded via a namespace (and not attached):
>  [1] annotate_1.42.1      AnnotationDbi_1.22.6 Biobase_2.20.1
>  [4] DBI_0.2-5            genefilter_1.46.1    geneplotter_1.42.0
>  [7] grid_3.1.1           lattice_0.20-29      locfit_1.5-9.1
> [10] RColorBrewer_1.0-5   RSQLite_0.11.2       splines_3.1.1
> [13] stats4_3.1.1         survival_2.37-7      XML_3.98-1.1
> [16] xtable_1.7-1         XVector_0.4.0
>
>
> --
> Nadia Kamal
> Bielefeld University
> Center for Biotechnology (Cebitec)
> Genome Research
> Universitätsstraße 27
> 33615 Bielefeld
> Germany
>
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