Hi,

I'm very new to RNA seq data analysis (so almost definitely doing something stupid!). I did an R course on DataCamp on Seq analysis, where I got some working code. I have a small RNA sequencing counts data set of mature miRNA counts (un-normalised). I have made the raw counts into a matrix with the row names and column names as the genes and sample name ( they definitely are not part of the matrix). I then have a data frame of the metadata for the samples.

this is my code up to the error message:

# download DESeq

if (!require("BiocManager", quietly = TRUE))
  install.packages("BiocManager")

BiocManager::install("DESeq2")

# Load library for DESeq2
library(DESeq2)

# Load library for RColorBrewer
library(RColorBrewer)

# Load library for pheatmap
library(pheatmap)

# Load library for tidyverse
library(tidyverse)

# read in data 
miRNA_counts <- read.csv("mature_counts.csv")

# Explore the first six observations 
head(miRNA_counts)

# Explore the structure of 
str(miRNA_counts)

# transpose data frame

miRNA_counts <- t(miRNA_counts)

colnames(miRNA_counts) <- miRNA_counts[1, ]

miRNA_counts <- miRNA_counts[-1,]

head(miRNA_counts)

# Create genotype vector
Volume  <- c("200", "100", "100", "200", "100", "200", "100")

# Create condition vector
condition <- c("Healthy", "Healthy", "Inflammatory", "Inflammatory", "Inflammatory", "Healthy", "Healthy")

# Create data frame
miRNA_metadata <- data.frame(Volume , condition)

# Assign the row names of the data frame
rownames(miRNA_metadata) <- c("PN0515_0008", "PN0515_0011", "PN0515_0004", "PN0515_0001", "PN0515_0010", "PN0515_0002", "PN0515_0005")

# check metadata and counts are in the same order

all (rownames(miRNA_metadata) == colnames(miRNA_counts))


# Create a DESeq2 object
dds_miRNA <- DESeqDataSetFromMatrix(countData = miRNA_counts ,
                                    colData = miRNA_metadata,
                                    design = ~ condition)

It is all fine up to the call of DESeqDataSetFromMatrix, where it throws an error message of "Error in DESeqDataSet(se, design = design, ignoreRank) : some values in assay are negative"

I have looked thorugh the entire dataset and there are no negative values anywhere. There are however, what i would consider weird spacing for the "0" values:

chr [1:947, 1:7] " 0" " 0" " 0" " 0" " 0" " 0" " 0" " 0" " 0" " 0" " 0" " 0" "0" ...

• attr(*, "dimnames")=List of 2 ..$ : chr [1:947] "hsa.let.7a.5p" "hsa.let.7a.3p" "hsa.let.7b.5p" "hsa.let.7b.3p" ... ..$ : chr [1:7] "PN0515_0008" "PN0515_0011" "PN0515_0004" "PN0515_0001" ...

what am I doing wrong here? I cant work it out!

What is the output of

below_zero <- apply(miRNA_counts  < 0, 1, sum) > 0
miRNA_counts[below_zero,,drop=FALSE]