Using DESeq2with Oxford Nanopore Technology Direct RNA Sequencing

Question

[uhlkatie]

I am trying to develop a workflow for performing differential expression analysis on long read direct RNA sequencing performed using the MinION from Oxford Nanopore Technology. The transcriptomics pipeline offered by ONT has a few bugs that still need to be worked out, so I am working out my own. Does anyone have any experience taking RNA-seq data from the MinION into DESEQ2 or a similar package? I started by using minimap2 (due to the fact that the parameters can be adjusted for nanopore technology) to generate a summarized experiment. I attempted to read in a table of metadata containing the list of fastq files and corresponding conditions into a data frame, but I received the error:

Error in colData<-(*tmp*, value = new("DataFrame", rownames = NULL, : nrow of supplied 'colData' must equal ncol of object

Perhaps I don't have the right understanding of what the metadata .csv file (I've also seen it referred to as a sample table) is supposed to look right. But that appears to be the one step that is preventing me from funneling my results into DESEQ2. If anyone has any advice or experience working with MinION data, I would really appreciate it.

Thanks!

Answer 1

[ATpoint]

This is not a Nanopore-specific problem. Your coldata has fewer rows (=entries) than you have samples. Given you had 11 samples with different treatments, this is how a coldata could look for these 11 samples, where NAME corresponds to colnames of the count table.

> coldata
                    NAME GENOTYPE STIMULUS FACTORIZED
1  KO_Ca_stimulated_rep1       KO       Ca          A
2  KO_Ca_stimulated_rep2       KO       Ca          A
3   KO_unstimulated_rep1       KO        U          B
4   KO_unstimulated_rep2       KO        U          B
5   KO_unstimulated_rep3       KO        U          B
6  WT_Ca_stimulated_rep1       WT       Ca          C
7  WT_Ca_stimulated_rep2       WT       Ca          C
8  WT_Ca_stimulated_rep3       WT       Ca          C
9   WT_unstimulated_rep1       WT        U          D
10  WT_unstimulated_rep2       WT        U          D
11  WT_unstimulated_rep3       WT        U          D

[uhlkatie]

Thanks so much! I finally got it to work!