Questions Regarding Ancestral Karyotype Reconstruction Using ODP With 7 Species

[aaannaw]

Dear ODP development team,

I hope this message finds you well. I am currently using ODP to investigate karyotype evolution within a genus for which we have chromosome-level genome assemblies for seven species. Our goal is to infer ancestral karyotypes and identify rearrangement events among these species.

I would like to ask several questions regarding the recommended workflow and configuration.

1. How should I choose species triplets for initial ALG reconstruction?

Your documentation suggests that steps 1–4 of the pipeline are best performed using species triplets to avoid loss of orthologs due to overly stringent filtering. However, I am not sure how I should select the triplets from the seven species in my dataset.

Our species tree is as follows (please see attached/tree structure below):

Is there a recommended strategy for generating triplets (e.g., selecting one representative per major clade, using sliding window triplets along the tree, etc.)?
Or is it acceptable to run all seven species together as I attempted?

2. Running all seven species simultaneously produces incomplete and cluttered ribbon plots

I ran the n-way pipeline with the following command:

snakemake -p --snakefile /data/01/user157/software/odp/scripts/odp_nway_rbh --cores 40

My config file includes all seven species at once. The pipeline completed successfully, producing the folders:

db

step0-blastp_results

step1-rbh

step2-groupby

step3-unwrap

step4-ribbon_diagram

Inside step2-groupby, I obtained:

ACAH_ACIL_ADIM_AKEM_AMIN_APER_ARUS.rbh.filt.groupby

And inside step3-unwrap:

ACAH_ACIL_ADIM_AKEM_AMIN_APER_ARUS.filt.unwrapped.rbh has 39 ALGs

However, the final ribbon plot:

ACAH_ACIL_ADIM_ADIM_AKEM_AMIN_APER_ARUS.ribbon.pdf

is extremely cluttered and seems to be cut off/unrendered in parts.

May I ask:

Should I adjust the species order or the chromosome order for each species?

3. Is an outgroup required for accurate ancestral karyotype reconstruction?

Given that all seven species belong to the same genus, would including an outgroup (e.g., from a sister genus) improve reconstruction of ancestral linkage groups and rearrangement polarity?
Or is ODP able to infer ancestral states without an outgroup?

4. Where can I find the configuration file example for odp_rbh_plot_mixing?

I would like to customize the mixing/plotting parameters, but I was unable to locate the example config file for:

odp_rbh_plot_mixing

Is there a sample config or documentation available for this script?

5. Full config file

For reference, here is the config file I used (attached or pasted below):

I would greatly appreciate any guidance you could provide on how to properly set up the analysis for seven species, how to construct triplets for ALG building, whether an outgroup is necessary, and how to control plot rendering for clarity.

Thank you very much for your time and for developing such a useful tool.

Best regards,
Na Wan