Question about variation in MEME-SEA results across gene sets

[Megan Barkdull]

Dear all, 

I have a question about the results I am getting from MEME-SEA. 

I am checking for motif enrichment in the promoters of four sets of genes:

1. Genes under positive selection in "foreground" species
2. Genes under positive selection in "background" species
3. Genes under relaxed selection in my species
4. Genes under intensified selection in my species

So I am running SEA four times, each time with promoters for a set of genes as the target set, and all other promoter sequences as the control set. Between target and control sets, sequences should have essentially identical lengths, because I just selected regions 2000bp upstream of all genes as putative promoters. 

I am concerned because, across runs, SEA detects very different numbers of matches to the same motif. For example, comparing the intensified selection results and the relaxed selection results, matches to nub are found in 0.8% and 0.2% of target and control sequences in that first analysis, but in 94.2% and 90.9% in the second analysis, even though both analyses use the same universe of promoter sequences. This seems biologically unlikely to me.

SEA is also calculating very different score thresholds between these two analyses (16.46 vs. 5.73), which I imagine contributes to this discrepancy. However, I am concerned that this indicates my results may not be super reliable. 

I'm happy to share my code, inputs, or output files if that is helpful. Any thoughts or input are really appreciated!

Megan Barkdull

[cegrant]

Hi Megan,

It would be super helpful if you could forward us examples of your SEA output. A good troubleshooting step would be to re-run your four sets of genes but allow SEA to use the default control sequences. By default SEA will use a shuffled version of the input sequences as the control, preserving both monomer and dimer frequencies. The problem with choosing your own control sequences is that the target and control sequences may have very different nucleotide frequencies. One issue that frequently crops up is that GC rich sequences may have spurious matches to GC rich motifs. 

You can attach files by clicking on the paper clip icon next to the "Post message" button.

[Megan Barkdull]

Thanks for your response! Hopefully my inputs have attached correctly and everything is playing nicely with my organization's security settings. The commands that I ran are as follows:

`/programs/meme-5.5.2/bin/sea --p ./14_SEAenrichment/relaxedSelectionpromotersUnderSelectionSequences.fasta --m. /14_SEAenrichment/JASPAR2022_CORE_insects_non-redundant_pfms_meme.txt --n ./14_SEAenrichment/relaxedSelectionpromotersNotUnderSelectionSequences.fasta --oc ./14_SEAenrichment/relaxedSelection/`

`/programs/meme-5.5.2/bin/sea --p ./14_SEAenrichment/intensifiedSelectionpromotersUnderSelectionSequences.fasta --m ./14_SEAenrichment/JASPAR2022_CORE_insects_non-redundant_pfms_meme.txt --n ./14_SEAenrichment/intensifiedSelectionpromotersNotUnderSelectionSequences.fasta --oc ./14_SEAenrichment/intensifiedSelection/`

`/programs/meme-5.5.2/bin/sea --p ./14_SEAenrichment/foregroundOnlypromotersUnderSelectionSequences.fasta --m ./14_SEAenrichment/JASPAR2022_CORE_insects_non-redundant_pfms_meme.txt --n ./14_SEAenrichment/foregroundOnlypromotersNotUnderSelectionSequences.fasta --oc ./14_SEAenrichment/foregroundOnly/`

`/programs/meme-5.5.2/bin/sea --p ./14_SEAenrichment/backgroundOnlypromotersUnderSelectionSequences.fasta --m ./14_SEAenrichment/JASPAR2022_CORE_insects_non-redundant_pfms_meme.txt --n ./14_SEAenrichment/backgroundOnlypromotersNotUnderSelectionSequences.fasta --oc ./14_SEAenrichment/backgroundOnly/`

I'll try your troubleshooting suggestion as well- thank you!

_____________________________

Megan Barkdull

She/her

Ph.D Candidate | Moreau Lab

NSF Graduate Research Fellow

Department of Ecology and Evolutionary Biology, Cornell University 

Website

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[cegrant]

Hi Megan, I'm afraid your files didn't seem to be attached. You can also email them to meme-...@uw.edu

[César Martínez]

Hi! I have a similar issue on my data. Have you resolved finally the question?

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[cegrant]

Hi Caesar,

Please start your own question with details about the problem you are having. If possible forward us copies of your input data and the exact command you used. Issues tend to be quite specific to the exact input data and options used, so piggybacking on other folks' questions generally isn't very useful.