Trouble with EdgeR DE (in Trinity)

[sjeschonek]

Hello everyone, I am very new to RNA-seq and trinity and command line, please bear with me!

I have successfully assembled my runs and am now trying to perform a differential expression between two samples using the edgeR method (described in the trinity manual). However, I keep erroring, and could use some insight on how to fix it.

[sjeschon@login001 ~]$ /gpfs/runtime/opt/trinity/2013-08-14/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix /gpfs/data/kmowry/sjescho                n/RNASeqResults/2transcripts.counts.matrix --method edgeR --output /gpfs/data/kmowry/sjeschon/RNASeqResults/edger0
Got 2 samples, and got: 3 data fields.
Header: assembly_trinity_15443687.isoforms.results      assembly_trinity_17707296.isoforms.results
Next: agalma/gene:21261.0/isoform:4     489.05  NA

$VAR1 = {
          'assembly_trinity_17707296' => 2,
          'assembly_trinity_15443687' => 1
        };
$VAR1 = {
          'assembly_trinity_17707296' => [
                                           'assembly_trinity_17707296'
                                         ],
          'assembly_trinity_15443687' => [
                                           'assembly_trinity_15443687'
                                         ]
        };
Samples to compare: $VAR1 = [
          'assembly_trinity_17707296',
          'assembly_trinity_15443687'
        ];

CMD: R --vanilla -q < 2transcripts.counts.matrix.assembly_trinity_15443687_vs_assembly_trinity_17707296.assembly_trinity_15443687.vs.assembly_tri                nity_17707296.EdgeR.Rscript
> library(edgeR)
Loading required package: limma
>
> data = read.table("/gpfs/data/kmowry/sjeschon/RNASeqResults/2transcripts.counts.matrix", header=T, row.names=1, com='')
> col_ordering = c(1,2)
> rnaseqMatrix = data[,col_ordering]
> rnaseqMatrix = round(rnaseqMatrix)
> rnaseqMatrix = rnaseqMatrix[rowSums(rnaseqMatrix)>=10,]
> conditions = factor(c(rep("assembly_trinity_15443687", 1), rep("assembly_trinity_17707296", 1)))
>
> exp_study = DGEList(counts=rnaseqMatrix, group=conditions)
> exp_study = calcNormFactors(exp_study)
Error in if (any(allzero)) x <- x[!allzero, , drop = FALSE] :
  missing value where TRUE/FALSE needed
Calls: calcNormFactors
Execution halted
Error, cmd: R --vanilla -q < 2transcripts.counts.matrix.assembly_trinity_15443687_vs_assembly_trinity_17707296.assembly_trinity_15443687.vs.assem                bly_trinity_17707296.EdgeR.Rscript died with ret (256)  at /gpfs/runtime/opt/trinity/2013-08-14/Analysis/DifferentialExpression/run_DE_analysis.p                l line 439.




WARNING: This EdgeR comparison failed...

Any help would be greatly appreciated!

[sjeschonek]

Not sure if the error is due to the NA in the table?

Error in if (any(allzero)) x <- x[!allzero, , drop = FALSE] :
missing value where TRUE/FALSE needed