MANUAL FOR PROGRAM MAKEINF

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

(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*

APPLYING MAKEINF TO YOUR SEQUENCES AND ALIGNMENTS

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