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I am doing a project on duplicated genes and I have a major difficulty on how to align sequences that use different genetic codes. I work with fasta files that contain sequences of protein coding genes, every fasta file includes genes from many species that are orthologs/paralogs. These genes even in the same species may use different genetic codes. My goal is to align the sequences, codon alignment [the process of codon alignment considers the reading frame of the translated protein and adjusts the nucleotide alignment so that the protein alignment stays in frame.] , and afterwards to create N-J trees. For the whole process I work on MEGA11. I cannot organize the species-genes into groups with common genetic codes because I need all of them in the tree. One option, to my mind at least, would be the alignment not in codon but in DNA,or another option is to align their protein sequences or lastly a third option could be the change of the sequences in order to get rid of the stop codons and afterwards to use one common code for everything, by doing that I know that I will lose some info but at least I can work.Do you have any idea how to overcome this obstacle? Do you agree with any of this options? Thank you in advance!

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    $\begingroup$ Can you say a little more about how diverse the organisms that you're studying are? Hominins? Carnivores? Mammals? Vertebrates? Eumetazoa? Eukarya? Scale matters! the more distantly related your organisms are (including divergent genetic codes), the more you would want to use a more stable data type such as protein sequence. $\endgroup$ Commented Dec 6, 2023 at 5:20
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    $\begingroup$ Why is the genetic code relevant? There are not that many genetic codes out there, and most differences are around stop codons as you point out, but if you are aligning nucleotides, it shouldn't really matter. What does "not in codon but in DNA" mean? And yes, aligning proteins would make sense, did you try that? $\endgroup$
    – terdon
    Commented Dec 6, 2023 at 10:24
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    $\begingroup$ @pippo1980 no, that's a different thing. There are a few different genetic codes used by some species, and even our own mitochondria. See ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi#SG2. $\endgroup$
    – terdon
    Commented Dec 7, 2023 at 9:38
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    $\begingroup$ ncbi.nlm.nih.gov/pmc/articles/PMC8629427/…. A computational screen for alternative genetic codes in over 250,000 genomes , here I got some background info, thanks @terdon $\endgroup$
    – pippo1980
    Commented Dec 7, 2023 at 14:48
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    $\begingroup$ My sense is that you are overthinking it- just use protein sequence! $\endgroup$ Commented Dec 12, 2023 at 4:53

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I am having a very similar issue, however mine are multiple sequences of the same specie. I have a mixture of DNA and RNA sequences so although it appears to aligning fine the proteins produced are not correct. I’m at a loss on how to fix this so if you managed to figure out what to do please let me know.

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