I have mtDNA sequences of the cox1 gene from various populations of one species. I want to perform an AMOVA allele frequency analysis via Arlequin. This allele frequency package however requires my sequences to have the same length.

  • Is it possible to use my sequences?
  • Do I have to align them first maybe?

The length difference is most likely due to indels/deletions. Do I have to use specific parts of the sequence for each gene of the subunit?

  • 1
    $\begingroup$ Please edit your question and give us some more details. Are you talking about protein sequences? DNA? mRNA? Arlequin can perform various types of analysis, what analysis are you trying to do? $\endgroup$
    – terdon
    Dec 19, 2022 at 10:14
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    $\begingroup$ Thanks, that helps. I don't know what you mean by subunit though, do you mean subunit of the gene's protein product, of cytochrome c oxidase? Or are you working on some sort of protein complex formed by the protein products of multiple genes? In any case, yes, you would very likely need to align the sequences first. Why not at least try that and see if it works? $\endgroup$
    – terdon
    Dec 19, 2022 at 10:46
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    $\begingroup$ Oh BTW @Nickmofoe the correct name for the gene is cox1. Please avoid using the old nomenclature of COI, this can create confusion. $\endgroup$
    – M__
    Dec 19, 2022 at 12:52
  • $\begingroup$ There are various workflows that do this that are easy to google, for example here. Can you clarify how these don't work for you? $\endgroup$ Dec 19, 2022 at 19:15

1 Answer 1


All that is needed is to align the sequences within a formal aligner like MAFFT or muscle. Then pass that through the PDGSpider converter. Arlequin should just take it from there.

Manual annotation of the alignment is normal e.g. seqoTron and is traditionally regarded as the best approach rather than do this algorithmically. Thus I'm suggesting (strongly suggesting) do this prior the PGDSpider conversion. If an alignment has not been performed Arlequin should throw an error.

If there are trailing gaps (indels or '-') at the beginning and end of the alignment Arlequin will/should ignore these. You can of course simply cut them out with the alignment editor (Mega X will also work for alignment editing). The important point is that Arlequin must correctly identify homologues its an allele frequency package and nothing more. If there are errors in the alignment Arlequin will believe there is population structure when it could be absent.

I can't honestly believe that Arlequin will count an indel '-' as an informative site. It should just ignore those sites, because it is a very respectable package that has been around for years and years.

Okay, I am now signing off for Christmas... with occasional exceptions.


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