Limma-voom can't find object

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1

Hey guys,

First time posting here. I’m running an RNA-seq analysis but I am stuck at the Limma-voom step.

Here’s the error I am getting:
Error in eval(ej, envir = levelsenv) : object ‘Control_Old’ not found
Calls: makeContrasts → eval → eval
Execution halted

I just genuinely can’t understand how it isn’t found. I double checked the spelling and everything.

2

Hi, did you ever work out the solution to this problem? I’m having the same issue.

Thanks!

3

Hi @EmWhat

This error comes up when the tool cannot build a data structure properly during processing. This is an R error, so R formatting requirements need to be met.

The tool form has examples/detailed help for each of these:

  1. At least two groups are input
  2. Each group contains at least two count inputs
  3. The same number of count files were input per group
  4. Labels for groups are all “oneword” or “one_word”. Alphanumeric characters plus (optionally) underscores.
  5. If you are inputting a matrix instead of individual count files, the same labeling rules would apply.
  6. Same for contrast inputs – full group labels separated by a dash “-”.
  7. Whitespace or any other characters will cause interpretation problems.

If you can’t find the problem, please post back either a shared history link or a screenshot of the job details page for one of the errored outputs (specifically, the portion of the log where the tool version and inputs/parameters are listed out). How to do both is explained here troubleshooting-errors

Let’s start there :slight_smile:

Related prior Q&A: edgeR error in galaxy

4

Hi @jennaj ,

I wanted to follow up on this here since I am having the same issue applying limma to a countMatrix and colData file for a test subset of my overall data. I’ve done the checklist based on your prior solution post.

Firstly, here is the simple workflow:
image

The countMatrix input data (image below) contains four samples

image
image1278×782 37.3 KB

The associated colData file is given in the image below, matching the filename/samplename to each group within the condition ‘Oxygen’, and being passed in as the factor list. It is a .csv that has been uploaded and tabularized using Convert CSV to tabular

image
image1307×236 19.3 KB

My workflow is set up such that the contrast is ‘Hypoxia-Normoxia’ .

Yet I still get the error:
Error in eval(ej, envir = levelsenv) : object ‘Hypoxia’ not found

I am comparing samples from two lanes, which makes the sample names prior to the first underscore identical (not sure if this contributes to the issue at all, but I’m passing them as separate sample names in the factor list). I will merge lane BAM files later and compare between different biological replicates across lanes as well to better harmonize file vs sample names.

Any insight is greatly appreciated!

5

Just following up on this… I had a bit of cognitive failure and was originally attempting to pass the data as separate count files (Hence the FileName column that was still present in the Factor List). I failed to update the column after consolidating the count files into a Matrix. Removing this has fixed this issue for me.

1 Like
6

Hi @James_jxcol Thank you for originally posting your details then posting back what worked! very helpful for everyone who finds this topic later on :slight_smile: