generate a common AnnotateMyID file from two different runs

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Hi all,
I have featureCounts file belonging from two different RNA-seq runs and analysis. I have 3 controls and 3 treated files. They have been analyzed till makinf the featurecounts.
The second group is a new run from new samples, also controls and treated, for which I got featurecounts as well.
However, the row order of the Entrez id is not the same, nor the total number of entrez. Hence, I cannot generate a common Anotated ID file with “AnnotateMyIDs” tool and therefore analyze them together with Limma

Is there any way to organize them in a way that I can analyze both runs together, hence to generate a single Annotated IDs file?

Thanks a lot for your help!

Best,
Lucy

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Hi @TheMisaXD

Different samples will always have slightly different expression, even from the same condition. Some samples will have expression for genes that others do not. That means the results from a tool like Featurecounts will have different “hits” once run through AnnotateMyIDs.

Now, you could also get different results if the reference data used was different, so be sure to double check that all samples were processed with the same data and parameters. This is really important. Rerun if you need to since the final result will have scientific problems if different files were used.

Have you seen our tutorials for this analysis pileline? These are linked from the bottom of the tool forms, or see the End-to-End tutorials here.

The first one ends with the creation of a count matrix using the FeatureCounts results. This merges together all of the results into a single table.

Then you can run AnnotateMyIDs on the count table, and get just one unified output for use with Limma.

All of those tutorials have workflows that you could decide to import and adjust to fit your data. That would allow you to run everything in a batch. It makes it easier to adjust small scientific parameters and quickly rerun, or to process more samples later on without all the tedious per tool work.

We have a guide here that includes some tips about reference data and related details → FAQ: Extended Help for Differential Expression Analysis Tools

I hope this helps and you can ask more questions! :slight_smile: