I am currently experiencing difficulties to run the DESeq2 analysis for finding DEGs for my GeoMx Data.

The place I am stuck at is the constructing of the DESeqDataSet object.

packageVersion(“DESeq2”)
‘1.44.0’
R version 4.4.1
The dataset was normalised by RUV4.

The tutorials I am following are:
▾https://lashlock.github.io/compbio/R_presentation.html
▾https://master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html#the-deseqdataset-object-sample-information-and-the-design-formula

So far the top 2 lines successfully ran, but the last part – “DEseqDataSetFromMatrix” function is not working with the tidy = TRUE.

From the tutorial, it states that the tidy=TRUE argument tells DESeq2 to output the results table with rownames as a first #column called ‘row.

Would it be okay to continue without the tidy = TRUE part?

Thank you in advance. It would be amazing if I could get through this and go on to the next step.

Code:

HNcountdata <- round(assays(HNspe_ruv)[["counts"]])
HNcoldata <- colData(HNspe_ruv)

HNdds <- DESeqDataSetFromMatrix(countData = HNcountdata, colData = HNcoldata, design=~biology, tidy = TRUE) 

Error
Error in .rowNamesDF<-(x, value = value) :
duplicate ‘row.names’ are not allowed

Dataset:
colData(HNspe_ruv)
DataFrame with 3 rows and 36 columns
SlideName ScanLabel ROILabel SegmentLabel
<character> <character> <character> <character>

Origin Instrument ID QCFlags AOISurfaceArea AOINucleiCount ROICoordinateX
<character> <logical><numeric> <numeric> <numeric>

ROICoordinateY RawReads AlignedReads DeduplicatedReads TrimmedReads StitchedReads
<numeric> <numeric> <numeric> <numeric> <numeric> <numeric>

SequencingSaturation SequencingSetID UMIQ30 RTSQ30
<numeric> <character> <numeric> <numeric>

ROIID SegmentID ScanWidth ScanHeight ScanOffsetX
<character> <character> <numeric> <numeric> <numeric>

ScanOffsetY Disease_status Regions sample_id lib_size countOfLowEprGene
<numeric> <character> <character> <character><numeric> <numeric>

percentOfLowEprGene biology ruv_W1 ruv_W2
<numeric> <character> <numeric> <numeric>

*I have intentionally deleted the content inside the table, which I hope will not be a problem to understand the structure of my dataset.

If I delete the tidy = TRUE,
it does run, but it comes up like this.

converting counts to integer mode
Warning: some variables in design formula are characters, converting to factors Note: levels of factors in the design contain characters other than
letters, numbers, ‘‘ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters ‘‘ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]