NG-NEW: A Modified Nei-Gojobori Method for Computing
                Synonymous and Nonsynonymous Distances

(c) Copyright March 1998 by Jianzhi Zhang and the Pennsylvania State University.
Permission is granted to copy this document provided that no fee is charged
for it and that this copyright notice is not removed.  NG-NEW is distributed
free of charge by

        Jianzhi Zhang
        Institute of Molecular Evolutionary Genetics
        and Department of Biology
        322 Mueller Laboratory
        The Pennsylvania State University
        University Park, PA 16802, USA

        Telephone: 814-8657030
        Fax: 814-8637336
        Email: zhang@imeg.bio.psu.edu

        Suggested citation:

        Zhang J, Rosenberg HF, Nei M (1998)  Positive Darwinian selection after
        gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci.
        USA 95:3708-3713.


1. Introduction
        NG-NEW is designed for estimating synonymous and nonsynonymous distances
between protein coding DNA sequences.  The method is modified from the original 
Nei and Gojobori (1986) method to take into account the transition bias.  The program
is written in C language and can be used on IBM PC compatible computers with the 
windows95 operating system.


2. Installation
        First make sure that the diskette you have received contains the
following files.

    ng-new.c         (source code)
    ng-new2.c        (source code)
    ng-new.exe       (executable file)
    ng-new2.exe      (executable file)
    manual           (this file)
    rnase.seq        (an example data file)
    outfile          (output file)

        To install NG-NEW on your computer's hard disk drive ("C" drive
given here, for example), you should create a directory where the files of
this package will be present.  To do this, type the following
                c:\md ng-new  (Enter)

        To copy the NG-NEW files onto your hard disk drive, insert the
floppy disk containing the programs into your floppy drive ("A" drive given
here, for example).  Then, enter the following command
                c:\copy a:*.* c:\ng-new\*.*  (Enter)

        The difference between the programs ng-new.exe and ng-new2.exe is that the
latter does not compute the covariance matrix, which makes large-data (>100 sequences)
analysis feasible.

3. Input file
        To use the program, you need an input file containing the protein coding
DNA sequences (see rnase.seq for an example).  This file begins with two numbers:
the number of sequences and the number of nucleotides per sequence (sequence length).
The second line will be the name of the first sequence, and the third line will be 
the first sequence, and so on.  Only A, G, C, T, a, g, c, and t are allowed.  Gaps
should be removed and sequences should be aligned beforehand. The sequences should 
only include protein-coding regions, with stop codons removed

4.  Notations.
R:  transition/transversion ratio.  R=0.5 means no transition bias.  Note that R is
    not the transition/transversion rate ratio (which is often denoted by kapa).
    Under Kimura's model, 2R=kapa. 
S:  number of synonymous sites of a sequence.
N:  number of nonsynonymous sites of a sequence.
s:  number of synonymous differences between two sequences.
n:  number of nonsynonymous differences between two sequences.
ps: p-distance (proportion) of synonymous difference.
pn: p-distance (proportion) of nonsynonymous difference.
ds: Jukes-Cantor distance of synonymous difference.
dn: Jukes-Cantor distance of nonsynonymous difference.
 
5.  Computation.

        To compute S, N, s, n, ps, pn, ds, and dn, type
                c:\ng-new\ng-new filename
        For example, to try the rnase.seq data, type
                c:\ng-new\ng-new rnase.seq

        You will be asked to input the transition/transversion ratio (R), which should
be estimated beforehand.  If you want to use the original Nei-Gojobori method, 
input R=0.5.
        The variances and covariances of distances are computed according to Ota and
Nei (1994).

5. Output file
        There are several output files with different formats.

outfile: this is most useful, including S, N, s, n, ps, pn, ds, dn, and variances.
sn.rst:  this file includes covariances, in addition to those quantities given in 
         outfile.
s.dis:   this file is used as an input file for bn-bs.exe.
n.dis:   this file is used as an input file for bn-bs.exe.

The files sn.rst, s.dis, and n.dis are generated only when ng-new.exe is used.