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MANUAL FOR PROGRAM MAKEINF
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INTRODUCTION
____________

MAKEINF (for "MAKE INFile") reads an amino acid or nucleic acid
alignment of the format of Jotun Hein's ALIGN program (MBE 1989,
6:649-668) and then outputs an aligned set of nucleic acid sequences in
the sequential format used by the PHYLIP package. Please note that if
you don't use ALIGN, you can easily edit your alignments so that they
can be used with MAKEINF.

WHEN do I want to use this program?

It is particularly useful for the analysis of protein-coding genes at
the nucleic acid level. It may also be used on nucleic acid sequences
which do not encode proteins. I wrote it because I analyze first plus
second position changes which result in amino acid replacements.
Editing such sequences can be extremely time-consuming when it is done
by hand.

a) Given an alignment, MAKEINF puts the gaps in the right places by
using the alignment (usually an amino acid alignment, but nucleic acids
are ok as well) as a template on which to align the corresponding
nucleic acid sequences. The resulting output file is directly PHYLIP-
readable.

b) If you have an amino acid alignment, the program asks you which
codon position you will analyze. There are five choices: first only,
second only, third only, first plus second, or all positions. The
program will only output the positions you want to analyze.

c) You can exclude as many sequences as you want. The program will only
output the sequences you want to analyze.

d) You can exclude blocks in which homology is uncertain. The program
will not output those positions.

e) For protein coding sequences, the progam allows you to convert first
positions of Leucine codons to a degenerate 'Y' (T or C), and those of
Arginine codons to a degenerate 'M' (A or C). This eliminates the noise
and the heterogeneity of rates which is introduced by silent changes in
these two classes of codons. If you analyze mitochondrial sequences, it
will only do this with first positions of leucine codons.

f) The program counts the number of A's, C's, G's, and T's, and also
Y's and M's, and allocates two-thirds of them to C, and one-third to T
or A, respectively, consistent with the number of codons encoding
leucine and arginine in the genetic code. It does this only if first
positions are included; if they are not, one can more easily have
PHYLIP calculate empirical base frequencies.


SUMMARY OF STEPS INVOLVED (illustrated with protein-coding sequences)
_________________________

Please note that if you don't use ALIGN, you can easily edit your
alignment so that it can be used with MAKEINF. See the next section for
the format of the alignment.

(1) Translate your protein-coding nucleic acid sequences.

(2) Prepare a file of the translated, unaligned, amino acid sequences
  for your alignment program.

(3) Apply ALIGN to the amino acid sequences, or another alignment
  algorithm. In the latter case you'll have to edit your alignment to
  make it MAKEINF-compatible.

(4) If desired, exclude sequences or blocks of uncertain homology.

(5) Prepare a file of the original nucleic acid sequences for program
  MAKEINF.

(6) Apply MAKEINF, which gives you a file with the nucleotide sequences
  in the sequential format used by PHYLIP. This file can be directly
  used as 'infile'.

(7) Apply DNAML (or another program of PHYLIP).



THE FORMAT OF THE ALIGNMENT FILE
________________________________

If you use ALIGN, you'll be familiar with the format of the alignment file.

If you don't use ALIGN, you can edit your alignment so that it can be used
with MAKEINF. This section tells you how to do that.

The following lines represent all the features in the alignment file which
are necessary for application of MAKEINF:

0 xen
1 wnt1
2 wnt2
3 wnt3
4 wnt4

ALIGNMENT

SGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKH---R-ESRGWVETLRAKYALFKPPTERDLVYY 66 3
SGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSDPPHLEPENPTHALPSSQDLVYF 70 0
SGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNR-ASRAELLRLEPEDPAHKPPSPHDLVYF 69 1
SGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQD---G-TGFTVA------NERFKKPTKNDLVYF 60 2
SGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRV---G-SSRALVPR----NAQFKPHTDEDLVYL 62 4
****   ***      *  *  *      *                                   ****


ENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCVF 123 3
EKSPNFCSPSEKNGTPGTTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTF 127 0
EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTF 126 1
ENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKF 117 2
EPSPDFCEQDIRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCHCRF 119 4
* **  *      *  **  * **  *   * *   *****           * * *


The features of the alignment format that are used as landmarks by
MAKEINF are the following:

(a) A list, of the numbers (starting with 0) and names of all
sequences, in order, without interrupting empty lines.

(b) The word 'ALIGNMENT' in capital letters, after the list of
sequences and before the alignment.

(c) The actual alignment with amino acids in capital letters. It
requires the following features:

 (i) It is in any number of blocks (the above example has two).

 (ii) EACH BLOCK NEEDS TO BE FOLLOWED BY AT LEAST ONE ASTERISK anywhere
   on the line immediately following the sequences. (ALIGN puts in these
   asterisks to indicate conserved residues.)

 (iii) NO EMPTY LINES MAY OCCUR WITHIN A BLOCK, but the number of lines
   between blocks does not matter. (Align sometimes puts certain characters
   between blocks; they do not interfere with MAKEINF.)

 (iv) The order of sequences within each block must be the same and all
   sequences listed on top must occur in the alignment.

 (v) Within the alignment, SEQUENCES ARE IDENTIFIED BY THEIR NUMBER.
   Note that there are two numbers at the end of each line. The first
   is the number of the last residue on that line. The second number is
   the ID number of the sequence on that line. (E.g., the first sequence
   in the above alignment is wnt3.) Both numbers must be there, but the
   value of only the second number (the sequence ID) is important. If you
   do not use ALIGN, and you want to leave out the first number, edit
   makeinf.c in the following manner: Delete the line
     fscanf( sourceali, "%d", &taxnum );     /* dumps the length number */
   in function 'findname'.

 (vi) The length of the sequences in each block must be the same (which they
   will be if the sequences are aligned) but the blocks can be of different
   lengths.

 (vii) GAP CHARACTERS ARE ALWAYS HYPHENS ('-'; ASCII 45).

 (viii) NO SPACES are allowed at the beginning of the lines with sequence
   information. They must begin with a capital letter, a hyphen, a square
   bracket, or a curly bracket.

 (ix) Any number of sequences may be excluded. Excluding sequences
   is done with CURLY brackets:

 SGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKH---R-ESRGWVETLRAKYALFKPPTERDLVYY 66 3
 {SGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSDPPHLEPENPTHALPSSQDLVYF 70 0}
 SGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNR-ASRAELLRLEPEDPAHKPPSPHDLVYF 69  1
 SGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQD---G-TGFTVA------NERFKKPTKNDLVYF 60  2
 SGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRV---G-SSRALVPR----NAQFKPHTDEDLVYL 62  4
 ****   ***      *  *  *      *                                   ****

 ENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCVF 123  3
 {EKSPNFCSPSEKNGTPGTTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTF 127  0}
 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTF  126  1
 ENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKF  117  2
 EPSPDFCEQDIRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCHCRF  119  4
 * **  *      *  **  * **  *   * *   *****           * * *

 In this example, sequence number 0 will not be written to the output file.
 Several sequences can be excluded according to the following format:

 SGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKH---R-ESRGWVETLRAKYALFKPPTERDLVYY 66 3
 {SGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSDPPHLEPENPTHALPSSQDLVYF 70 0
 SGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNR-ASRAELLRLEPEDPAHKPPSPHDLVYF 69 1}
 SGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQD---G-TGFTVA------NERFKKPTKNDLVYF 60 2
 SGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRV---G-SSRALVPR----NAQFKPHTDEDLVYL 62 4
 ****   ***      *  *  *      *                                   ****

 ENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCVF 123 3
 {EKSPNFCSPSEKNGTPGTTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTF 127 0
 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTF 126 1}
 ENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKF 117 2
 EPSPDFCEQDIRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCHCRF 119 4
 * **  *      *  **  * **  *   * *   *****           * * *

 or, if they are interspersed, like this:

 SGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKH---R-ESRGWVETLRAKYALFKPPTERDLVYY 66 3
 {SGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSDPPHLEPENPTHALPSSQDLVYF 70 0}
 SGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNR-ASRAELLRLEPEDPAHKPPSPHDLVYF 69 1
 SGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQD---G-TGFTVA------NERFKKPTKNDLVYF 60 2
 {SGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRV---G-SSRALVPR----NAQFKPHTDEDLVYL 62 4}
 ****   ***      *  *  *      *                                   ****

 ENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCVF 123 3
 {EKSPNFCSPSEKNGTPGTTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTF 127 0}
 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTF 126 1
 ENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKF 117 2
 {EPSPDFCEQDIRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCHCRF 119 4}
 * **  *      *  **  * **  *   * *   *****           * * *

 KEEP TRACK OF HOW MANY SEQUENCES ARE COMMENTED OUT, SINCE THE PROGRAM WILL
 ASK YOU FOR THE NUMBER OF SEQUENCES TO BE ALIGNED.

 (x) Any number of positions in which alignment is uncertain can be excluded.
   This is done with SQUARE brackets. In the above example, the gapped
   region in the first block ought to be omitted:

 SGSCEVKTCWWAQPDFRAIGDFLKDKYDSASEMVVEKH[---R-ESRGWVETLRAK]YALFKPPTERDLVYY 66 3
 {SGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSDPPHLEPENPTHALPSSQDLVYF 70 0}
 SGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNR[GSNR-ASRAELLRLEPE]DPAHKPPSPHDLVYF 69  1
 SGSCTLRTCWLAMADFRKTGDYLWRKYNGAIQVVMNQD[---G-TGFTVA------]NERFKKPTKNDLVYF 60  2
 SGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRV[---G-SSRALVPR----]NAQFKPHTDEDLVYL 62  4
 ****   ***      *  *  *      *                                   ****

 ENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCVF 123  3
 {EKSPNFCSPSEKNGTPGTTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTF 127  0}
 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTF  126  1
 ENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKF  117  2
 EPSPDFCEQDIRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCHCRF  119  4
 * **  *      *  **  * **  *   * *   *****           * * *

 Note that a sequence that is excluded need not get square brackets.


THE FORMAT OF THE NUCLEOTIDE FILE
_________________________________

The format of the file with the nucleotide sequences is the same as
that used by ALIGN. THE ORDER OF THE SEQUENCES IN THIS FILE MUST
CORRESPOND TO THE NUMBERING OF THE SEQUENCES IN THE ALIGNMENT. (I.e.,
in the above example, the order of nucleotide sequences is xen, wnt1,
wnt2, wnt3, wnt4.) Obviously, they have to be the sequences from which
the amino acid translation was derived. The nucleotide sequences must
correspond exactly to the amino acid sequences used for the amino acid
alignment. No extra or missing nucleotides are allowed. If the program
misses nucleotides, it will crash and give you an error message. If it
sees additional nucleotides it will give you a warning. Here is the
first sequence of the above alignment in correct format (the length of
each line is irrelevant):

>
xen
TCAGGATCCTGCTCCCTCAGGACGTGCTGGATGCGGCTTCCCCCCTTCCGTTCAGTTGGG
GATGCTTTGAAGGATCGTTTTGATGGAGCCTCTAAAGTGACCTACAGCAACAATGGCAGC
AATCGATGGGGTTCTCGCAGTGACCCACCTCACCTAGAACCTGAAAACCCCACACATGCT
CTGCCATCATCCCAGGATCTTGTCTATTTTGAGAAGTCTCCTAACTTCTGCAGCCCTAGT
GAAAAGAATGGAACTCCTGGAACCACAGGGCGAATATGTAACAGCACTTCATTGGGACTA
GATGGATGTGAACTCTTGTGCTGTGGTAGAGGATACCGGAGTCTGGCTGAAAAAGTCACT
GAACGGTGCCATTGCACATTT*


The salient features of this format are the 'GREATER THAN' symbol > ,
the NAME ON THE LINE FOLLOWING IT, the SEQUENCE (in capital or
lowercase characters), and the TERMINATION-SYMBOL ('*').
THESE FEATURES ARE ESSENTIAL.



APPLYING MAKEINF TO YOUR SEQUENCES AND ALIGNMENTS
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Compile makeinf.c with your favorite C compiler and execute it. The
program is semi-interactive in that it asks you a bunch of questions,
one by one. The user is prompted first for the names of the files to be
used, then for the number of sequences in the alignment, and then for
various options.

The following lines are what the screen of your computer looks like after
you answer all questions; this example is specific to the example files
provided with the program and this manual.


Alignment file to be read:      ex.ali
Nucleotide file to be read:     ex.nuc
Destination file to be written: infile

Total number of sequences in alignment: 5
Number of sequences to be used:         4
Nucleic acid or Protein coding sequence?          (n/p): p
Nuclear or mitochondrial genetic code?            (n/m): n

Enter a number between 1 and 5, for the
   codon position you wish to analyze:
   1 for first, 2 for second, 3 for third
   4 for first plus second, 5 for all.            (1-5): 4
Conversion of first positions to degenerate base? (y/n): y
Use nAmes or nUmbers as identifiers?              (a/u): a



Let's go through this one by one:

The program starts by writing 'Alignment file to be read:   ', i.e. it asks
you for the name of the file which holds the amino acid alignment. The user,
in this case, specified the name 'ex.ali', and hit ; ex.nuc holds
the nucleotide sequences; infile is the name of the file to which the
nucleotide alignment will be written. 5 is the number of sequences in
the alignment, but the number of sequences to be used is only 4 since
one (number 0, or 'xen') is commented out. In this example, we are dealing
with protein coding sequences (hence the 'p') of the nuclear code ('n').

Since we are dealing with protein coding sequences, the next question is
about which positions we want to use. Option 4 means: first plus second
positions. As we want to eliminate silent changes in first positions of
arginine and leucine codons, we anser yes ('y'); and we want to have the
program use the names ('a') in the output file.
When you've entered all these options, the program bounces the following
back at you:

Nucleotide sequences in:                 ex.nuc
Amino acid alignment source:             ex.ali
Nucleotide alignment destination:        infile
First plus second codon positions will be used.
L and R 1st positions will be converted to Y and M.
Names will be used to identify sequences.

Frequencies of A, C, G, T:
   0.26529   0.24032   0.27189   0.22250

The last two lines appear if you are using first positions of protein
coding sequences, and you requested that first positions of leucine
and/or arginine codons be converted to their degenerate base. When you use
PHYLIP, you should input these numbers, rather than use empirical base
frequencies, especially if silent positions in your sequences are not at
compositional equilibrium.

That's it. If you have questions that this manual does not answer, send
e-mail to

arend@mendel.berkeley.edu

Yours,

Arend Sidow