I'm interested in removing variation in my data using RUVseq. I'm following the tutorial/vignette and was able to generate the estimated factors of unwanted variation using the RUVg function. Now I want to incorporate them into my differential expression analysis using DESeq2.

Here is what I have:

set_NHP_RUVg <- RUVg(set_NHP, empirical, k=1)
pData(set_NHP_RUVg)
       key_IP_mf.Treatment        W_1
1M_IP                Day03 -0.2389338
2M_IP                Day03  0.3677343
3M_IP                Day03 -0.1828536
4M_IP                Day10 -0.2924691
5M_IP                Day10  0.3641969
6M_IP            Uninjured -0.3050515
7M_IP                Day20  0.1791624
8M_IP                Day20  0.3791267
9M_IP            Uninjured  0.3179415
11M_IP               Day20 -0.3741618
12M_IP           Uninjured -0.2146920

dds_RUVg <- DESeqDataSetFromMatrix(countData = counts(set_NHP_RUVg),
                              colData = pData(set_NHP_RUVg),
                              design = ~ W_1 + key_IP_mf$Treatment)

Error in DESeqDataSet(se, design = design, ignoreRank) : all variables in design formula must be columns in colData

But the two elements of my design are the two columns in my colData:

pData(set_NHP_RUVg)
       key_IP_mf.Treatment        W_1
1M_IP                Day03 -0.2389338
2M_IP                Day03  0.3677343
3M_IP                Day03 -0.1828536
4M_IP                Day10 -0.2924691
5M_IP                Day10  0.3641969
6M_IP            Uninjured -0.3050515
7M_IP                Day20  0.1791624
8M_IP                Day20  0.3791267
9M_IP            Uninjured  0.3179415
11M_IP               Day20 -0.3741618
12M_IP           Uninjured -0.2146920

sessionInfo( )
R version 4.2.2 (2022-10-31)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Monterey 12.6.2

Matrix products: default
LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] DESeq2_1.38.3               RColorBrewer_1.1-3          zebrafishRNASeq_1.18.0      RUVSeq_1.32.0              
 [5] edgeR_3.40.2                limma_3.54.2                EDASeq_2.32.0               ShortRead_1.56.1           
 [9] GenomicAlignments_1.34.1    SummarizedExperiment_1.28.0 MatrixGenerics_1.10.0       matrixStats_0.63.0         
[13] Rsamtools_2.14.0            GenomicRanges_1.50.2        Biostrings_2.66.0           GenomeInfoDb_1.34.9        
[17] XVector_0.38.0              IRanges_2.32.0              S4Vectors_0.36.2            BiocParallel_1.32.6        
[21] Biobase_2.58.0              BiocGenerics_0.44.0

I'm just missing something. But I'm also following the vignette exactly.

Not familiar with the package, but why the …$Treament in the formula when it’s key_IP_mf.Treatment?