AFRC TRAINING MANUAL - CHAPTER 15
                   15.  PHYLOGENY INFERENCING
                                
15.1.     Starting Phylip
     
The  PHYLogeny  Inferencing Package  is  not  part  of  GCG,  and
comprises  some  30  programs.  It  takes  data  in  sequence  or
"discrete  data" (numeric) form. You should make some  effort  to
understand the theory behind these programs if you use them.  The
following  examples  take a multiple sequence alignment,  convert
it  to PHYLIP format, then calculate a distance matrix. Trees are
derived  by  two different methods: Neighbor-Joining  and  Fitch-
Margoliash.

To start up the phylip package:
   
   $ Seqprogs
   $ Phylip

Note  the  list of program names. Documentation can be found  in
the directory PHYLIP_NOTES.

15.2.     MSF to Phylip
     
RNA.MSF is the output from a PILEUP of bacterial ribosomal  RNA.
Use  the  READSEQ program to convert to PHYLIP format, then  use
the editor to change any dots in the sequence to dashes.
   
   $ Readseq -a rna.msf -format=phylip -output=rna.phy
   $ Edit rna.phy

15.3.     The DNADIST program
     
DNADIST calculates pairwise distances between sequences,  taking
into  account (for DNA) transitions and transversions. NB: There
are four different methods for doing this in DNADIST!
   
   $ dnadist
   
        PHYLIP version 3.5c - running DNADIST
   
   Input file name (* DNADIST.DAT *) ? : rna.phy
   Output file name (* RNA.OUT *) ? : rna.dist
   Submit as batch job (* No *) ? :
   Nucleic acid sequence Distance Matrix program, version
   3.53c
   
   Settings for this run:
     D  Distance (Kimura, Jin/Nei, ML, J-C)?  Kimura 2-
   parameter
     T        Transition/transversion ratio?  2.0
     C   One category of substitution rates?  Yes
     L              Form of distance matrix?  Square
     M           Analyze multiple data sets?  No
     I          Input sequences interleaved?  Yes
     0   Terminal type (IBM PC, VT52, ANSI)?  ANSI
     1    Print out the data at start of run  No
     2  Print indications of progress of run  Yes
   
   Are these settings correct? (type Y or letter for one to
   change)
   y

Examine the distances file RNA.DIST
15.4.     The NEIGHBOR and FITCH programs
     
The  file  PHYLIP_NOTES:DISTANCE.DOC summarises  four  distance-
based   methods  for  nucleic  and  protein  sequences.   Fitch-
Margoliash  using  "the  PHYLIP search  method"  and  Neighbour-
Joining do not assume rate constancy across lineages. The  UPGMA
and  KITSCH programs assume rate constancy across lineages.  The
FITCH and Neighbour-joining methods are equally preferred,  with
NJ the faster program.
   
   $ Neighbor
   
        PHYLIP version 3.5c - running NEIGHBOR
   
   Input file name (* NEIGHBOR.DAT *) ? : rna.dist
   Output file name (* RNA.OUT *) ? : neighbor.out
   Treefile name to WRITE (* NEIGHBOR.TREE *) ? :
   
   Submit as batch job (* No *) ? :
   
   Neighbor-Joining/UPGMA method version 3.5
   
   Settings for this run:
   
     N       Neighbor-joining or UPGMA tree?  Neighbor-joining
     O                        Outgroup root?  No, use as outgroup
   species  1
     L         Lower-triangular data matrix?  No
     R         Upper-triangular data matrix?  No
     S                        Subreplicates?  No
     J     Randomize input order of species?  No. Use input order
     M           Analyze multiple data sets?  No
     0   Terminal type (IBM PC, VT52, ANSI)?  ANSI
     1    Print out the data at start of run  No
     2  Print indications of progress of run  Yes
     3                        Print out tree  Yes
     4       Write out trees onto tree file?  Yes
   
   Are these settings correct? (type Y or the letter for one
   to change)

The output file NEIGHBOR.OUT gives a phylogenetic tree.

Use the same distance data for FITCH:
   
   $ fitch
   
        PHYLIP version 3.5c - running FITCH
   
   Input file name (* FITCH.DAT *) ? : rna.dist
   Output file name (* RNA.OUT *) ? : fitch.out
   Treefile name to WRITE (* FITCH.TREE *) ? :
   
   Submit as batch job (* No *) ? :

Accept the defaults and examine the output file FITCH.OUT.

Do  the  trees produced by the two programs differ, or are  they
the same?

The  programs DRAWGRAM and DRAWTREE can be used to  display  the
TREE files.
RETREE can be used to rearrange the nodes on the trees.