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To identify homologous genes, I performed tBLASTn analyses against my genome of interest resulting in sequences of a closely related species. Using this method, I found two (and in some cases up to five copies) of my different genes of interest in the target genome.

How can I determine if these gene copies correspond to true duplication events?

I aligned the newly found sequences to the reference genes and I found good alignments in more than one region (20% - 50% identity), but I am unsure if this is enough to consider them as duplicates.

Is there a threshold to help me determine if they are indeed duplications? How can I distinguish between a true duplication event and a pseudogene or a paralogous gene? The target genome belongs to a polyploid organism, although the genome assembly type is listed as haploid.

Any help will be greatly appreciated.


Thank you all very much for your responses, I am now beginning to understand how to find out if my genes are duplicated.

I performed some of the analyses that you suggested and I would very much appreciate your input on the results.

First, I followed @terdon's advice and I took the best hit for one of the query (source) genes in the target genome and I used this sequence as a query against the query (source) genome, i.e., I performed a reciprocal BLAST analysis (in my case it was a reciprocal BLASTp analysis). The first hit was my original query gene, which had an E-value of 0.0. For other sequences the reciprocal BLASTp hits were more variable, ranging from 3e-42 to 2e-154, but the first hit was always the query sequence. I also looked at the protein domains and they are conserved, i.e., both the query and the target sequences have the same domains. As for the exonic structure, it is fairly similar, for e.g., the query has 12 exons while the target sequence has 10. Exons are similar in length. So, in summary, when the potential duplicates from my target genome are blasted against the original source genome, the best hit is the original query sequence.

As for @zhanf's suggestion, I did a basic phylogenetic analysis using Clustal Omega (neighbour-joining tree without distance corrections) using four sequences: the original query sequence and the three gene copies (seq_5-1, seq_5-2 and seq_5-3) that I found in the target genome. This is the result:

enter image description here

I also compared each of the sequences against the original query:

  • seq_5-1 vs query: 34% identity (query length 780 aa, seq_5-1 length 400 aa)
  • seq_5-2 vs query: 42% identity (query length 780 aa, seq_5-2 length 580 aa)
  • seq_5-3 vs query: 40% identity (query length 780 aa, seq_5-3 length 490 aa)

So in summary, these sequences would appear to be paralogues, right?

Again, thank you very much for your help, I greatly appreciate it!

Leah

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    $\begingroup$ I am not sure if the phrase "true duplication" would be correct, a pseudogene could very well be the result of a duplication prior to losing its function. If the question would be if some of the newly identified orthogonal genes are pseudogenes, you might want to check the length of the resulting ORFs from the orthogonal genes, I would expect strikingly shorter ORFs for pseudogenes (depending on the time of the duplication and the selection pressure on the species). $\endgroup$ – haci Aug 25 at 20:48
  • $\begingroup$ Homologues vs. paralogues ... you've got to be quite serious about the question for someone else to describe this in detail. Are you familiar with concerted evolution? $\endgroup$ – Michael G. Aug 25 at 22:56
  • $\begingroup$ I don't have enough reputation to comment, but you could also look at where they are located in the genome. duplicated genes are often arranged in a tandem array (right next to each other), although not always. If the genes are right next to each other in the genome, and they share a high sequence identity, they are likely duplicate genes. A much more specific way to tell, would be to make a phylogeny of the all of the supposed gene copies from the species you know. Depending on how they group together you may be able to tell if they are paralagous or separate genes. $\endgroup$ – zhanf Aug 26 at 20:43
  • $\begingroup$ Just to mention its easy to solve, but we'd need your cooperation. Understanding the gene and the organism would be a help. $\endgroup$ – Michael G. Aug 26 at 23:41
  • $\begingroup$ Thanks for your answer @Leah. The BLAST hits may not be the same gene, because thats alot of distance at 34-40% identity. The "duplication" event would not be what I would call a paralogue and I suspect the genes have different functions. The duplication event could have occurred (probably did occur) before the species has evolved, in which case I personally wouldn't consider that paralogy, but it depends at what taxonomic level you are looking at (would have to be pretty major taxonomic grouping). $\endgroup$ – Michael G. Aug 30 at 5:39
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You might want to have a look at What is the difference between orthologs, paralogs and homologs?. Paralogs are duplicates by definition, and whether a gene is a pseudogene or still active has no bearing on whether it comes from a duplication.

In any case, to figure out what's going in in your target genome, you should use the target's genes. So, first get the best hit for each of your query genes in the target genome. Then, to investigate its evolutionary history, use that sequence (the one from the target genome) as query against the target genome. That will let you assess how similar the target's hits are to one another and that's how you can attempt to figure out their evolutionary history.

Note that 20% identity is very low for closely related species. While there is no golden threshold, I would expect recent duplication events (and in closely related species, a duplication event that is in one genome and not the other will be recent) to produce much more similar sequences. In any case, you can't do this based on sequence identity alone.

Are the protein domains of the gene products conserved? Is the exonic structure of the gene conserved? You should also try taking the putative duplicate of geneA from genome2 and blast it against genome1 (your original source genome). Is the best hit the geneA from genome1, the query you used to find the putative duplicate in genome2? Or is there a better hit for it against another gene?

These are all questions to investigate and take into account when reconstructing the evolutionary history. You can't just look at identity and use some threshold to decide this sort of thing.

If you blast the potential dupicate from your target genome against your original source genome, is the best hit the duplicate?

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I don't have enough reputation to comment but if a sequence is duplicated then there has obviously been a duplication event. Doesn't matter how scientists call it now.It can be older or more recent but it's yet a duplication.

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