Neighbor Joining method in Phylip

To carry out phylogenetic analysis it is important to begin with an alignment in which all gap-containing positions have been removed.

CLUSTAL 2.0.12 multiple sequence alignment


zf_12a1_a       VADLVFLVDGSWSVGRENFRFIRSFIGA--
zf_12a1_b       KADLVFLIDGSWSIGDDSFAKVRQFVFS--
hs_22a1         HYDLVFLLDTSSSVGKEDFEKVRQWVAN--
hs_a11          YMDIVIVLDGSNSIYP--WVEVQHFLINIL
zf_a11          YMDIVIVLDGSNSIYP--WNEVQDFLINIL
                  *:*:::* * *:    :  :: ::    

• This can be accomplished using the gap strip tool.

• There is a bug in the process but the gap-free alignment can be loaded into clustalX, the the save sequences as "Phylip" format function can be used.

        5  26
zf_12a1_a  VADLVFLVDGSWSVGRFRFIRSFIGA
zf_12a1_b  KADLVFLIDGSWSIGDFAKVRQFVFS
hs_22a1    HYDLVFLLDTSSSVGKFEKVRQWVAN
hs_a11     YMDIVIVLDGSNSIYPWVEVQHFLIN
zf_a11     YMDIVIVLDGSNSIYPWNEVQDFLIN

• The correct alignment can be created with a text editor. Another version of it is in your clustal/phylip folder called tiny_nogap.phy

• enter the phylip folder:

cd /home/USERNAME/clustal/phylip/

• The first step to to create the bootstrap replicates. This is done with the phylip application seqboot.

seqboot

• when prompted, provide the name "tiny_nogap.phy".

• At the interactive menu:

  • change option r to 10 for 10 replicates. NOTE: normally the number used is 1000 for neighbor

  • press Y to accept changes

  • enter 111 as random number seed.

cat outfile

• Move outfile to tiny_nogap.seqboot

mv outfile tiny_nogap.seqboot

• The next step is to calculate the distances between the sequences in each replicate. This is done with protdist

protdist

• when prompted, provide the name "tiny_nogap.seqboot".

• At the interactive menu:

  • change option m to d for datasets and 10 for number of replicates.

  • press Y to accept changes

cat outfile

• Rename the outfile

mv outfile tiny_nogap.protdist

• Build the neighbor-joining tree for each distance matrix wth neighbor:

neighbor

• when prompted, provide the name "tiny_nogap.protdist".

• At the interactive menu:

  • change option m to 10 for number of replicates.

  • enter 111 for random number seed

  • press Y to accept changes

cat outfile
cat outtree

• Rename the output:

mv outtree tiny_nogap.neightree
mv outfile tiny_nogap.neighout

• Create a consensus version of the outtree file with consense:

consense

• when prompted, provide the name "tiny_nogap.neightree".

• At the interactive menu, default settings are ok so type "Y"

cat outfile
cat outtree

• Rename the output:

mv outtree tiny_nogap.neigh.constree
mv outfile tiny_nogap.neigh.consout

• Draw the tree with drawtree:

drawtree

• when prompted for a file enter tiny_nogap.neigh.constree

• when prompted for a fontfile, enter:

/net/n3/data/Teaching/IAP_2010_day3/clustal/font1

NOTE: a series of different fonts are available within the phylip installation directory

• At the interactive menu, select option V, then N to turn off preview. Then option Y

• This results in a postscript plot of the tree. rename it to:

mv plotfile tiny_nogap.tree.ps

• The contents of this file can be viewed with photo software like but it looks like this:

• The bootstrap values can be viewed in the file tiny_nogap.neigh.constree. Or graphicall by opening this file with NJ-plot. With branch lengths selected, the view looks like this: