1. What files do I need to run OrthoFinder?

    A single FASTA file containing protein sequences for each species.

  2. How many species can OrthoFinder handle?

    The latest version of OrthoFinder can handle thousands of species. It does this using the linear species addition framework, whereby orthogroups are first computed for a small core set, and then the remaining species are assigned without requiring an all-vs-all search.

  3. When should I use the linear species addition method?

    We recommend that you use the core-assign framework when you have too many species to do a regular OrthoFinder run in a reasonable time. That will depend on your computing resources, but as a rough guide:

    • If you are running >32 cores, use core-assign if analysing >100 species.
    • If you are on a personal computer running <32 cores, use core-assign if analysing >32 species.
  4. How many core species should I pick?

    It depends on how many species you are analysing (see response to question 3).

    • If you have >100 species, we recommend that you pick 64 core species.
    • If you have <100 species, we recommend that you pick 16 core species.

    Try to choose a set that covers a broad range of phylogenetic diversity across your species.

  5. How can I make OrthoFinder run faster?

    You can give OrthoFinder more CPU cores with the -t and -a commands.

  6. How is the species tree built?

    OrthoFinder infers a species tree using multi-copy gene families. OrthoFinder relies on Astral-Pro III and STAG to infer the species tree from orthogroups created during the initial BLAST and clustering process, these Orthogroups are converted into gene trees using a chosen tree inference method. The root of the unrooted species tree is identified using STRIDE which identifies well supported duplication events across the tree in order to correct place the root.

  7. Can I add my own species tree?

    Yes, you can supply a Newick tree with the -s option.

  8. Can I use a different gene-tree inference or alignment tool?

    Yes, just add the relevant command to the config.json file. You can then point OrthoFinder towards your config file using --config path/to/user/config.json, and then select your options using the -M and -T flags.

  9. What changed between OrthoFinder v2 and the new v3?

    The current version of OrthoFinder has several major changes compared to version 2:

    • •
      New workflow for scalability:
      The --core --assign workflow uses SHOOT to create profiles for previously computed orthogroups and adds new genes to these orthogroups without requiring a costly all-versus-all sequence search. Genes that cannot be assigned using SHOOT are analysed using the standard OrthoFinder workflow.
    • •
      Phylogenetic Hierarchical Orthogroups:
      OrthoFinder now defines orthogroups at every node in the species tree, significantly increasing accuracy and enabling clade-specific orthogroup analysis.

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