Phylogenetic Tree Construction using PHYLIP software

Step1: Download and install Phylip software from http://evolution.genetics.washington.edu/phylip/getme.html

Step2: Do multiple sequence alignment of the sequence of interest using ClustalX software. In clustalx software, choose "save as phylip" option. After MSA, you will be saved three output file".aln", ".dnd", and ".phy"  files.

Note: While doing clustalx, please note that the first header lines or organism names or protein names in fasta sequence file should be unique. If same or repetive names or gap between each fasta sequences occurs then clustalx will not run properly.

Step4: Copy ".phy file in C:/Program files/Phylip/exe/ folder

Step3: In C:/Program files/Phylip/exe/ folder, open bootstrap option. Now drag and drop"
".phy" phylip file, (calling it infile) Do 100 replicates. Give Random seed number: 3 (must be odd)
click Y to all the default parameters.

Step4. Open DnaDist  (call infile, if you want, the previous outfile ) and change dataset as MULTIPLE DATASET, telling you did 100 replicates

Step5:  Run neighbourjoining  (call infile, if you want, the previous outfile ), and set multiple dataset to 100. you can set a root.

 Now you have 100 trees in the outtree file.
you can already open this file with treeview, adding the extention .ph to the file.

Step6:  Create a consensus tree: Run Consensus calling intree the outtree from NJ.

Step7. See the outtree.ph (i.e, fourth file that generated after running neighbourjoining) in Treeview.

Notes: in treeview from Tree Menu set: Internal edge label to see the bootstrap values.
            The "real tree" is the first of the 100 created by NJ, the consensus tree is useful to know how reliable are the branches and the nodes.