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.
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).