I am trying to figure out how to calculate the dn/ds ratio of a set of genes which have 'fragmented' into two or more genes spanning the same 'reference' gene sequence. In some cases, this is due to indels, in others SNPs result in one or more stop codons along the coding region. I am using the full-length coding sequence as a reference - is it possible to calculate the ratios over two or more genes, if not - is it possible to normalise the ratios from analysing gene by gene?
I have attached an alignment file with coding regions outlined to give you an idea of what i am trying to analyse.
Hi Aileen,
if I understand your question correctly, your full-length gene has fixed a SNP (punctual SNP or Indel) that has either frame shifted your coding sequence generating stop codons or has introduced a stop codon in the middle of your coding sequence. In either case, I would say that the resulting sequence no longer codifies for a functional protein--that is, when using dN/dS throughout your protein coding sequence, this value should approximate 1 (the rate at which a pseudogene would evolve). I guess the easiest way to analyze the entire thing in one go is by removing the stop codon/s and running the scripts (PAML or DNAsp, etc depending on the nature of your data). You do not need to normalize anything because dN/dS is already normalized and hence is valid to compare different genes regardless the rate at which they fix mutations: In reality you are trying to measure selection pressures by estimating the number of amino acid replacing nucleotides per unit time (with time being approximated through the estimate of dS).
Hope this helps, otherwise let me know
Mario
Hi Aileen,
if I understand your question correctly, your full-length gene has fixed a SNP (punctual SNP or Indel) that has either frame shifted your coding sequence generating stop codons or has introduced a stop codon in the middle of your coding sequence. In either case, I would say that the resulting sequence no longer codifies for a functional protein--that is, when using dN/dS throughout your protein coding sequence, this value should approximate 1 (the rate at which a pseudogene would evolve). I guess the easiest way to analyze the entire thing in one go is by removing the stop codon/s and running the scripts (PAML or DNAsp, etc depending on the nature of your data). You do not need to normalize anything because dN/dS is already normalized and hence is valid to compare different genes regardless the rate at which they fix mutations: In reality you are trying to measure selection pressures by estimating the number of amino acid replacing nucleotides per unit time (with time being approximated through the estimate of dS).
Hope this helps, otherwise let me know
Mario
This fragmented gene sequences needs first to be aligned with the reference sequence. The Dn/Ds ratio is used to determine the presence of a pressure that are being imposed to determined sequence in comparison with another sequence, and, because of that, both need to be in the same reading fase. But I recommend you to perform the analyses also in two blocks, comparing the evolution of each of your fragmented genes separately with the "block" of the reference sequence corresponding. This way you can know if this fragmentation event altered the Dn/Ds ratio on this two genes
Has your reference gene been split to 'fragmented' genes by evolution? I'm not sure if the fragmentation could have technical reasons, e.g. sequencing errors. - If evolution has split the genes it is reasonable to ask for the evolutionary dynamics of the parts separately. In a gene coding for a multiple-domain protein, for example, the different gene regions may have very different evolutionary dynamics. A structural/catalytic domain probably is under negative selection, an unstructured loop may evolve neutrally, and an extracellular loop might even be subject to positive selection. So, I recommend to analyze the fragmented genes independently
Thank you all for your answers! Looks like I will have to stick it through going gene by gene.
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