So I have been trying to construct a phylogenetic tree based on a collection of ITS sequences of a library of fungal isolates I cultured. However, after constructing it on MEGAX with my aligned sequences (ensuring they were of similar length/no N's) there is one group of 3 isolates that are placed between 2 other branches that share the same class (the 3 isolates between are all closely related to each other, but belong to a different taxonomic class than the two on either side of them). However, despite trying various models and different types of trees, I find they only become less accurate regarding the known relatedness of the various genera (based on their taxonomic ranks).
Has anyone had similar issues with constructing phylogenetic trees from nucleotide sequences? I acknowledge it may be somewhat flawed to try and model the evolutionary descent of these isolates based on ITS sequences, but any advice would be appreciated.
Thanks in advance.
It would be better if you can provide pictures of the tree. However, in this case, you may need to make sure if your ITS sequences are the same region of ITS from the database. Which primer did you use or did you design it by yourself? And also make sure whether you use forward or reverse sequence in your alignment seqs.
I used the ITS4-F 5′-TCCTCCGCTTATTGATATGC-3′ and ITS1F-R 5′-CTTGGTCATTTAGAGGAAGTAA-3′ primers for all samples. Also, it was the COntig0 of all my sample forward and reverse sequences that I aligned in Mega, would this make a difference?
Many thanks
Some fungi of the same genus have high nucleic acid similarity in certain functional genes, and it is important to ensure that you select the appropriate sequences for phylogenetic tree analysis. In addition, you can combine results from TEF 1α sequences for analysis.
Best wish！
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