# 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](http://www.hiv.lanl.gov/content/sequence/GAPSTREEZE/strip_ready.html) 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](http://pbil.univ-lyon1.fr/software/njplot.html). With branch lengths selected, the view looks like this: