"Only 2 columns of data: need at least 3" error when trying to use Limma for RNA-Seq data

Hello there, I am trying to generate some DE tables using RNA-Seq data in Limma but I am getting this error message:

Warning message:
In Sys.setlocale(“LC_MESSAGES”, “en_US.UTF-8”) :
OS reports request to set locale to “en_US.UTF-8” cannot be honored
Error in plotMDS.default(y, top = top, labels = labels, pch = pch, cex = cex, :
Only 2 columns of data: need at least 3
Calls: plotMDS → plotMDS.DGEList → plotMDS → plotMDS.default

Is there something missing in my dataset? I can share my history if needed.

Welcome, @auerbachsd

The error message is directly from the underlying R Bioconductor tool, and it is reporting that it expected a 3 column file for one of the inputs, but the file submitted only had two columns.

Where to look for examples:

  1. Scroll down on the tool form to the Help section. I see two possible three column input files. If you are using one of those, compare your file(s) to the examples and you’ll probably spot the problem.

  2. Scroll down a bit more to find links to any GTN tutorials that happen to include this tool (not always available for every tool, but this one has the link). This will have example data, and hands-on training, and workflow(s) that you can use as a template when running your own analysis.

  3. Try a search at the original tool authors resources – links are usually also included in the Help section of tool forms, but a plain internet search can also find related resources/discussions.

Limma has general usage help at the Bioconductor support site, and in their tool vignettes. This means that a search at the support site with parts of the error message might show some more clues about how the data is processed inside the tool, which can help with organizing your experiment. The overall processing will be the same as when running the tool in Galaxy versus directly (the wrapper in Galaxy is parsing your data and sending it to the same underlying tool for manipulations, then writing the results back inside Galaxy). Sometimes worth a look …but come back here for most Q&A, especially if this is new to you. https://support.bioconductor.org/

Please give those a review first. If you get stuck, you can share back your work for more help. How-to is in the banner at this site, or see directly →

Hi jennaj, I made the change and it works perfectly! Thank you for your help!

1 Like