**20**wrote:

Hi!

This is my first post here and I'm new to all of this in general, so I hope I can do this correctly!

I have a specific question about incorporating the results from RUVSeq(specifically RUVg) into the time series analysis of DESeq2.

My RNASeq experiment is a time course (4 points) of two different strains, similar to the fission yeast dataset used in the DESeq2 example. We used the Thermofisher ERCC RNA Spike In mix for use as a negative control to normalize our samples.

Following the example in the RUVSeq documentation (RUVSeq: Remove Unwanted Variation from RNA-Seq Data, compiled 2017), I used RUVg to calculate the estimated factors of unwanted variation ("W_1") as done in the example and shown below.

set1 <- RUVg(set, spikes, k=1)

In an example of a two sample comparison in DESeq2, the documentation includes the the factors of unwanted variation as such:

dds <- DESeqDataSetFromMatrix(countData = counts(set1), colData = pData(set1), design = ~ W_1 + x) dds <- DESeq(dds) res <- results(dds)

Where `W_1`

= the factors of unwanted variation and `x`

= the two groups (control and treated).

I would like to use the factors of unwanted variation in the setting of a time course with a likelihood ratio test. In the DESeq2 documentation (RNA-seq workflow: gene-level exploratory analysis and differential expression, 2017) the following is given using the fission yeast dataset.

ddsTC <- DESeqDataSet(fission, ~ strain + minute + strain:minute) ddsTC <- DESeq(ddsTC, test = "LRT", reduced = ~ strain + minute) resTC <- results(ddsTC)

My question is how I should incorporate the unwanted variation factors without interfering with the parameters for a time course analysis. Is it as simple as adding it as the first component of the design as I've written below?

ddsTC <- DESeqDataSet(fission, ~ W_1 + strain + minute + strain:minute) ddsTC <- DESeq(ddsTC, test = "LRT", reduced = ~ W_1 + strain + minute) resTC <- results(ddsTC)

I'd appreciate any input on how to correctly incorporate these factors of unwanted variation with a time course. If someone could explain or point me to some information to help me understand how the design components interact with the analysis I'd appreciate it as well. Thanks!

**830**• written 20 months ago by staceyborrego •

**20**