MANUAL FOR PROGRAM MAKEINF

INTRODUCTION

WHEN do I want to use this program?

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-codingsequences)

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
* **  *      *  **  * **  *   * *   *****           * * *

(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:

1. It is in any number of blocks (the above example has two).
2. 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.)
3. 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.)
4. The order of sequences within each block must be the same and all sequences listed on top must occur in the alignment.
5. 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'.
6. 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.
7. GAP CHARACTERS ARE ALWAYS HYPHENS ('-'; ASCII 45).
8. 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.
9. 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.
10. 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

>
xen
TCAGGATCCTGCTCCCTCAGGACGTGCTGGATGCGGCTTCCCCCCTTCCGTTCAGTTGGG
GATGCTTTGAAGGATCGTTTTGATGGAGCCTCTAAAGTGACCTACAGCAACAATGGCAGC
AATCGATGGGGTTCTCGCAGTGACCCACCTCACCTAGAACCTGAAAACCCCACACATGCT
CTGCCATCATCCCAGGATCTTGTCTATTTTGAGAAGTCTCCTAACTTCTGCAGCCCTAGT
GAAAAGAATGGAACTCCTGGAACCACAGGGCGAATATGTAACAGCACTTCATTGGGACTA
GATGGATGTGAACTCTTGTGCTGTGGTAGAGGATACCGGAGTCTGGCTGAAAAAGTCACT
GAACGGTGCCATTGCACATTT*

APPLYING MAKEINF TO YOUR SEQUENCES AND ALIGNMENTS

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

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