Hello,

I have a quick question. I have used Deseq before but I did not need to run a time-series on my data before and so I am a little stumped. For my data, I have sampled over time, from the same subplots which are nested within a plot for an entire year. So my data looks as follows: with 9 sites (3 unburned controls and 6 burned), each plot has 4 subplots embedded within them.

![Sample Data[1] [1]: https://ibb.co/m9dT7X9

I have run the following code and it works, but when I enter the site, which is where the samples are imbedded I get an error stating Error in checkFullRank(modelMatrix). Please see code below.

*I have looked online and though this site and I am honestly not sure how to proceed.*

- I was originally following this tutorial from Deseq under time series, but again I am stumped at the nested part.
- I want to contrast for treatment between the different time points,so any help or guidance is really appreciated.

**This is the code I have as of right now**
dds<-phyloseq*to*deseq2(physeq, design = ~ Treatment + TimePoint)
gm*mean = function(x, na.rm=TRUE){
exp(sum(log(x[x > 0]), na.rm=na.rm) / length(x))
}
geoMeans<-apply(counts(dds), 1, gm*mean)
dds<-estimateSizeFactors(dds, geoMeans = geoMeans)
dds<-DESeq(dds, test= "LRT", fitType="local", reduced = ~ Treatment)

**#Look at results**
res<- results(dds, contrast = list(c("T1",c("T2" ,"T3","T4","T5","T6","T7","T8","T9"))
res<- res[order(res$padj, na.last=NA), ]
alpha<-0.01
signif<-res[(res$padj < alpha), ]
sigif<-cbind(as(sigtab, "data.frame"), as(tax_table(dds)[rownames(sigtab), ], "matrix"))
signif

** This code works but when I add Site as a variable I can no longer run the code: and instead, I get the following error:**
dds<-phyloseq

*to*deseq2(physeq, design = ~ Treatment + TimePoint+ Site )

** I've tried this but not sure if this is right and or how to to get Deseq to understand it.**
1. Create a nested column nested column and (link here)
2. Is this the proper way of doing it how do I add it and have the Deseq function read it.

*Please help, I have spent all day on this and have no idea how to proceed.*

I know how to add it in a normal regression, is it added in the same manner in Deseq?

DESeq2 is simply based on linear models. You are getting the same model you would get with

`lm`

. They both use`model.matrix`

to generateX.