Dear fellows,
I'm working on algal 18S rRNA and currently at the stage of phylogenetic tree construction. Among the sequences one has persistently failed to cluster with any of the sequences producing significant alignments from NCBI search. This is happening despite a percent identity of approximately 98, E value 0 and query coverage of 100% between the query and subjects. Surprisingly, sequences relatively lower in terms of these score parameters don't show similar recalcitrance. I'm using maximum likelihood algorithm in MEGA X, and I have tried different options such as partial deletion, complete deletion, use all sites, bootstrap replications as low as 50 etc. but all to no avail. What would be the possible explanation to this and how would this be resolved?
Thank you
If I understand you correctly, you have one sequence of 18S rRNA that is not grouped with the others.
Some observations will be necessary before beginning the stage of phylogenetic analysis.
First, was the 18S rDNA gene fully or partially sequenced?
If different regions were partially sequenced between different sequences, they may be the source of the problem.
Second: Are all the sequences used in the global alignment in the same direction? Foward or reverse? It is very common that eventually one of the sequences used in the global alignment is in a different direction from the others, which can also generate this type of problem.
Another option is to change something in the phylogenetic analysis. The maximum likelihood method is powerful and capable of detecting small differences between the analyzed sequences. Perhaps testing other construction methods like pacimony or some distance-based method will solve the problem.
Thank you for your useful contributions Mathew A Beale . I didn't do anything like that; in fact this is a new knowledge to me. I will check that out. After reading your comment, I visited the multiple sequence alignment file and I found some substitutions (up to 17 bases) along the length of my sequence (1227 bases long). Could that be some sort of substitution mutation or it is too early to tell?
Thank you once again
Thank you Rodrigo Pereira . First of all, we used full sequence. Second, we added sequences of the same sense/direction (+/+ strand). As for trying less sensitive methods, what would be your assessment of a tree built using any of those please? I understand that experts tend to show preference towards maximum likelihood method. Please correct me if I'm wrong.
Thank you
Dear Abdullahi Inuwa , each phylogenetic construction method has advantages and disadvantages. In addition, it is not always possible to use the same method for all situations. Personally, I use the values of boostrap to check whether the built tree is reliable or not. However, the solution indicated by @Mathew A Beale to use seaview is very interesting.
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