ClustalX2 guide tree

Question

[m.taheri]

I am going to align a bunch of protein sequences using ClustalX2. This software first builds a guide tree from pairwise distances between sequences and uses that tree for multiple alignment of sequences. I need to know the order of sequences which are chosen for alignment via the guide tree. ClustalX2 by default uses neighbor-joining algorithm to create the guide tree and stores the tree in a .dnd file. But when I visualize the tree it seems it is unrooted because it has three branches in its first level.

The following picture shows my tree that ClustalX2 stores in a .dnd file:

1- How do I know this tree is rooted or unrooted?

2- If this tree is unrooted how ClustalX2 uses it as guide tree? As far as I know the guide tree should be rooted.

Thanks.

Answer 1

[h.mon]

ClustalX is a graphical interface, I suppose the alignment program is actually ClustalW. According to ClustalW help, the estimated tree is unrooted:

4) To calculate a tree, use option 4 (DRAW TREE NOW). This gives an UNROOTED tree and all branch lengths. The root of the tree can only be inferred by using an outgroup (a sequence that you are certain branches at the outside of the tree .... certain on biological grounds) OR if you assume a degree of constancy in the 'molecular clock', you can place the root in the 'middle' of the tree (roughly equidistant from all tips).

edit:

You are correct, the above snippet from the manual describes getting a phylogenetic tree from a multiple sequence alignment. However, it turns out the method used by clustalw to get a rooted tree is the same as described above: placing the root at a position where the means of the branch lengths on either side of the root are equal. See the ClustalW paper: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

[m.taheri]

The guide tree should not be confused with the phylogenetic tree we will construct later. The guide tree is entirely based on the pairwise alignments, and is used to guide the construction of the multiple alignment. The phylogenetic tree is based on multiple alignment of sequences. My question is about the guide tree, not phylogenetic tree.

[h.mon]

You are indeed correct. I have updated my answer.

[m.taheri]

Thank you for your answer but my question is: Where is the root of the guide tree I have shown in my post? It seems it is an unrooted tree.