DeSeq2 "some values in assay are negative"

Question

[Morris_Chair]

Hello everyone, I'm back again with RNA-seq data analysis and I encountered an error that seems to be common for the work that I'm doing

dds <- DESeqDataSetFromMatrix(countdata, coldata, design=~condition)

Error in DESeqDataSet(se, design = design, ignoreRank) : some values in assay are negative

I tried this solve in this way:

mat <- countdata[,-1]

rownames(mat) <- countdata[,1]

message: non-unique values when setting 'row.names': ‘0’, ‘1’, ‘2’, ‘3’, ‘4’, ‘5’, ‘6’, ‘7’, ‘8’, ‘9’, ‘10’, ‘11’, ‘12’, ‘13’, ‘14’, ‘15’, ‘16’, ‘17’, ‘18’, ‘19’, ‘20’, ‘21’, ‘22’, ‘23’, ‘24’, ‘25’, ‘26’, ‘27’, ‘28’, ‘29’, ‘30’, ‘31’, ‘32’, ‘33’, ‘34’, ‘35’, ‘36’, ‘37’, ‘38’, ‘39’, ‘40’, ‘41’, ‘42’, ‘43’, ‘44’, ‘45’, ‘46’, ‘47’, ‘48’, ‘49’, ‘50’, ‘51’, ‘52’, ‘53’, ‘54’, ‘55’, ‘56’, ‘57’, ‘58’, ‘59’, ‘60’, ‘61’, ‘62’, ‘63’, ‘64’, ‘65’, ‘66’, ‘67’, ‘68’, ‘69’, ‘70’, ‘71’, ‘72’, ‘73’, ‘74’, ‘75’, ‘76’, ‘77’, ‘78’, ‘79’, ‘80’, ‘81’, ‘82’, ‘83’, ‘84’, ‘85’, ‘86’, ‘87’, ‘88’, ‘89’, ‘90’, ‘91’, ‘92’, ‘93’, ‘94’, ‘95’, ‘96 [... truncated]duplicate 'row.names' are not allowed In addition: WarningError in .rowNamesDF<-(x, value = value) :

but I can't find duplicated rows...

I accept any suggestion and thank you for help

[ATpoint]

head(countdata)

head(coldata)

[Morris_Chair]

Hi ATpoint,

here is the result

 > head(countdata)
                   SRR5088824_starReadsPerGene.out.tab.x SRR5088825_starReadsPerGene.out.tab.x
ENSG00000000003.10                                    70                                    70
ENSG00000000005.5                                      0                                     0
ENSG00000000419.8                                     83                                    83
ENSG00000000457.9                                     62                                    62
ENSG00000000460.12                                    39                                    39
                   SRR5088834_starReadsPerGene.out.tab.x SRR5088835_starReadsPerGene.out.tab.x
ENSG00000000003.10                                    70                                    70
ENSG00000000005.5                                      0                                     0
ENSG00000000419.8                                     83                                    83
ENSG00000000457.9                                     62                                    62
ENSG00000000460.12                                    39                                    39
                   SRR5088842_starReadsPerGene.out.tab.x SRR5088843_starReadsPerGene.out.tab.x
ENSG00000000003.10                                    70                                    70
ENSG00000000005.5                                      0                                     0
ENSG00000000419.8                                     83                                    83
ENSG00000000457.9                                     62                                    62
ENSG00000000460.12                                    39                                    39
                   SRR5088844_starReadsPerGene.out.tab.x SRR5088859_starReadsPerGene.out.tab.x
ENSG00000000003.10                                    70                                    70
ENSG00000000005.5                                      0                                     0
ENSG00000000419.8                                     83                                    83
ENSG00000000457.9                                     62                                    62
ENSG00000000460.12                                    39                                    39
                   SRR5088860_starReadsPerGene.out.tab.x SRR5088861_starReadsPerGene.out.tab.x
ENSG00000000003.10                                    70                                    70
ENSG00000000005.5                                      0                                     0
ENSG00000000419.8                                     83                                    83
ENSG00000000457.9                                     62                                    62
ENSG00000000460.12                                    39                                    39


 getOption("max.print") -- omitted 1 rows ]


> > head(coldata)

   condition 
SRR5088824_starReadsPerGene.out.tab.x responsive
SRR5088825_starReadsPerGene.out.tab.x responsive
SRR5088834_starReadsPerGene.out.tab.x responsive
SRR5088835_starReadsPerGene.out.tab.x responsive
SRR5088842_starReadsPerGene.out.tab.x responsive
SRR5088843_starReadsPerGene.out.tab.x responsive

I shorted the countdata list

Thank you

[ATpoint]

Please format data and code with the code button, not as a quote, it's messy and unreadable that way. The counts must be a numeric matrix. Rows being genes and columns being samples.

[Morris_Chair]

even if I convert the data.frame as matrix doesn't help

countdata<-as.matrix(countdata)
dds <- DESeqDataSetFromMatrix(countdata, coldata, design=~condition)
>Error in DESeqDataSet(se, design = design, ignoreRank) : 
>some values in assay are negative

[Marco Pannone]

Did you make sure that:

all(rownames(coldata) == colnames(count_matrix))

?

[Morris_Chair]

Hi Marco, It comes as "TRUE "

all(rownames(coldata) == colnames(countdata))
>[1] TRUE

thanks

[Marco Pannone]

Have you tried the workaround suggested here?

[Morris_Chair]

yes I did it but without success :(

[Marco Pannone]

When you want to reassign rownames, try this way:

mat <- countdata[,-1]
names_countdata <- countdata[,1]
rownames(mat) <- make.names(names_countdata, unique=TRUE)

[Morris_Chair]

Hi Marco I think I found the error, the problem is on my merged files which duplicates values in the same boxes and does some more errors..

Thank you for your answers :)