Overview of xalign

---

xalign has all of the following attributes which make it a very powerful yet relatively easy to use program:

1. Multiple alignments of up to 500 sequences at one time.
2. User has control of the alignment, the ability to include specific insights or knowledge.
3. Secondary structure information can be included for any or all of the sequences.
4. Multiple alignment has a consensus sequence.
5. Detailed pairwise alignments can be printed.
6. The ability to change the amino acid similarity matrix, gapping penalties, even the order in which the sequences are aligned.
7. The user can anchor the multiple alignment at places where he/she sees fit (eg, what would the alignment look like if amino acid X and Y have to line up).
8. The user can ensure that certain amino acids are not broken up by a gap within an alignment.

Copyright and Acknowledgements

---

Copyright (C) 1994 - No portion of this program may be incorporated into other programs or sold for profit without express written consent of the authors.

Basic Sequence Input File for xalign

---

Each input sequence must contain these minimum attributes:

1. A right angle bracket ">" signals the beginning of a sequence.
2. An ID code for the sequence which is alphanumeric character string (1-8 characters in length). Use as descriptive a name as possible.
3. The sequence name and other details on the remainder of the line.
4. The amino acid sequence on all subsequent lines in one letter code notation (upper or lower case).

The number of amino acid codes per line does not matter, however, it is easier to check your input for correctness if you decide on a constant number such as 50. Also, blanks are ignored if found in the amino acid sequence. Alternative amino acid code meanings such as 'B', 'X', and 'Z' are acceptable input but they will have no scoring value during the alignment process (unless the amino acid scoring matrix is changed).

Here is an example input file:

	>CaM Calmodulin - Drosophila melanogaster (1-148)
	ADQLTEEQIA EFKEAFSLFD KDGDGTITTK ELGTVMRSLG QNPTEAELQD
	MINEVDADGN GTIDFPEFLT MMARKMKDTD SEEEIREAFR VFDKDGNGFI
	SAAELRHVMT NLGEKLTDEE VDEMIREANI DGDGQVNYEE FVTMMTSK

	>TnC Troponin C, cloned chicken skeletal muscle (1-162)
	ASMTDQQAEA RAFLSEEMIA EFKAAFDMFD ADGGGDISTK ELGTVMRMLG
	QNPTKEELDA IIEEVDEDGS GTIDFEEFLV MMVRQMKEDA KGKSEEELAN
	CFRIFDKNAD GFIDIEELGE ILRATGEHVI EEDIEDLMKD SDKNNDGRID
	FDEFLKMMEG VQ

Here is an example input file with secondary structure information included:

	>CaM Calmodulin - Drosophila melanogaster (1-148)
	ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQD
	ccccchhhhhhhhhhhhhhccccccbbbhhhhhhhhhhcccccchhhhhh
	MINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFI
	hhhhhccccccbbbhhhhhhhhhhhhhcccchhhhhhhhhhhhcccccbb
	SAAELRHVMTNLGEKLTDEEVDEMIREANIDGDGQVNYEEFVTMMTSK
	bhhhhhhhhhhcccccchhhhhhhhhhcccccccbbbhhhhhhhhhcc

	>TnC Troponin C, cloned chicken skeletal muscle (1-162)
	ASMTDQQAEARAFLSEEMIAEFKAAFDMFDADGGGDISTKELGTVMRMLG
	cccchhhhhhhhhcchhhhhhhhhhhhhhccccccbbbhhhhhhhhhhcc
	QNPTKEELDAIIEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAN
	cccchhhhhhhhhhhccccccbbbhhhhhhhhhhhhhcccccccchhhhh
	CFRIFDKNADGFIDIEELGEILRATGEHVIEEDIEDLMKDSDKNNDGRID
	hhhhhccccccbbbhhhhhhhhhhhccccchhhhhhhhhhhccccccbbb
	FDEFLKMMEGVQ
	hhhhhhhhhhcc

Advanced Sequence Input File for xalign

---

The following section explains how the user can enter specific knowledge into the alignment process.

Sometimes xalign will insert gaps into an alignment where you think are not correct. You could change any of the various gapping penalties in the parameter file but this will likely change your entire alignment (which you may already be happy with). To prevent the program from breaking up a certain section of amino acids, just type asterisks above those amino acids. Because the program ignores blanks in the input sequence, the other amino acids without asterisks must get some kind of default character. In this case, use a "-" or dash character.

Here is an example of a sequence input file which has this amino acid clustering:

         >unkn1 unknown protein mouse 
         -------------******-------------------------------
         SRTEYDPLKFWPITHYCPHSARKDTYPERFYANMPKLDNQGPLSTYPLST
         cchhhhhhhhhchhhhhhhccccccccccbbbbccccccccccccchhhh
         ---------------
         QWPIIVDTASATLMS
         hhcbbbbbbcccccc

Another potentially useful tool is to be able to anchor a certain amino acid in one sequence to a certain amino acid in another sequence. One can imagine a scenario where a user knows that two amino acids line up but because of remote homology, xalign can't understand the significance of that particular match.

To implement this anchoring procedure, the user specifies a number between 1-5 above the first amino acid to anchor in the first sequence. The user then specifies that same number above the second amino acid in the second sequence.

Here is an example of anchoring one amino acid in one sequence to another amino acid in another sequence:

         >unkn1 this protein unknown for mouse
         ---------1----------------------------------------
         SRTEYDPLKFWPITHYCPHSARKDTYPERFYANMPKLDNQGPLSTYPLST
         cchhhhhhhhhchhhhhhhccccccccccbbbbccccccccccccchhhh
         ---------------
         QWPIIVDTASATLMS
         hhcbbbbbbcccccc

         >unkn2 some other protein
         ---------1----------------------------------------
         SRSELDPLKFMPLPITYCGHSAREATYPERDDANMPKLENSTGPLQTYPL
         ------------------
         LSYQCPIIVDTAKHLLNS

Output Files

---

1. program version and current date
2. list of alignment parameters used
3. (optional) pairwise alignments for each pair of sequences
4. final multiple alignment with consensus sequence

The xalign program was developed to handle the majority of alignment requests in a reasonable manner. Compromising the relatively straight forward algorithm for special classes of alignments of probable nature seemed beyond the intent of the program. Since the tools are available for the user to correct the errors, let he/she use them.

Alignment Parameters

---

The "xalign.parms" parameter file contains default settings for gap penalties, amino acid similarity and also some useful output options. The program looks for an "xalign.parms" file in the current directory, if one does not exist it uses the xalign.parms file found in the directory where you installed the program. Users who are interested in changing some of default settings in order to get better alignments may want to copy the above file to their current directory and try various changes.

An example parameter file is shown below.

****************************************************************
XALIGN Parameters
****************************************************************

Enter the file which contains the amino acid similarity
scoring matrix and the various gap penalties.
This file contains a lower triangular matrix which indicates
the similarity score between two amino acids. The matrix  is
of  a  general nature and may not reflect the specific needs
of the user (for example, a user who finds that cystines are
not lining up with cystines may want to increase the score
for such a match). 

XALIGN:SCOR_MATRIX  $INSTALL/lib/xalign/wtm.rbo

----------------------------------------------------------------

Indicate if pairwise alignments should be printed. This  can
greatly increase the output however it often contains useful
information.  Enter "1" for printing and "0" for not  printing.

XALIGN:PRINT_PAIRWISE 0

----------------------------------------------------------------

The following is an offset added to the percent homology score
of a pairwise alignment if secondary structure is known for a
given sequence.  In general, any sequence which has secondary
structure known should be given precedence over those that do not
when determining the order of sequences in the multiple alignment.

An offset of "0" means the order of the sequences in the multiple
alignment algorithm is based only on primary sequence homology.

Range: MIN_STRUCT_BIAS <= x <= MAX_STRUCT_BIAS

XALIGN:STRUCT_BIAS 100

----------------------------------------------------------------

As more and more sequences are added to the multiple alignment,
the cumulative effect of all the amino acid scoring starts to
dwarf the gapping penalties.  Therefore we probably want to
increase the gap and gap size penalties by a given percent for
each sequence added.

For example, this number should be adjusted up if too many gaps
are found in the final sequences of the multiple alignment. 

Range: MIN_INC_GAP_PEN <= x <= MAX_INC_GAP_PEN

XALIGN:INC_GAP_PEN 30   

----------------------------------------------------------------

Indicate if the pairwise sequence percent homology table should
be printed.

This table allows the user to quickly compare pairwise sequence
alignments in terms of percent homology.
Enter "0" for not printing and "1" for printing.

Range: 0 <= x <= 1

XALIGN:PRINT_PCT_HOM 1

----------------------------------------------------------------

Indicate the order of the sequences in your multiple alignment
output.

To have sequences printed in the order that the program ranks
them (for adding them to the multiple alignment), enter "0".

If you prefer to have the sequences printed in the order that
they were entered into the program, enter "1".

Range: 0 <= x <= 1

XALIGN:PRINT_ORDER 0

----------------------------------------------------------------

Following the multiple alignment, a consensus sequence is
printed.  At what percent identity is a consensus reached for
a given amino acid at a given position?

Enter an integer from 0 - 100 to represent this threshold.

Range: 0 <= x <= 100

XALIGN:CONSENSUS_PCT 70

----------------------------------------------------------------

Do not change these parameters, they were added for the sake of
compatibility with other packages that use the low level 
xalign engine.

XALIGN:DATA_FORMAT	1
XALIGN:DATA_FILE    x
XALIGN:SEQ_SELECT   1

Default Weighting Matrix

---

#
# rbo amino acid similarity matrix
#
# This matrix indicates how important it is to line up any given
# pair of amino acids (eg, aligning a cysteine with another
# cysteine is worth "n" points). 20 refers to number of amino 
# acid character codes. 

KEY:WT_PMAT		20
   A  C  D  E  F  G  H  I  K  L  M  N  P  Q  R  S  T  V  W  Y
A 10
C  2 17
D  2  2 10
E  2  2  6 10
F  1  0  0  0 12
G  2  2  2  2  0 10
H  3  1  2  2  3  2 10
I  2  2  0  0  6  0  1 10
K  3  2  3  3  0  2  3  0 10
L  2  2  0  0  6  0  0  4  0 10
M  2  2  0  0  4  0  2  4  1  8 10
N  2  0  6  4  0  3  3  0  7  0  1 10
P  2  0  3  0  0  2  3  0  2  0  0  2 10
Q  2  0  4  6  0  1  4  1  4  0  1  6  2 10
R  2  2  2  2  0  2  4  0  7  0  1  2  2  4 10
S  5  2  2  2  0  2  1  0  2  0  0  4  3  2  3 10
T  3  2  2  2  0  2  2  2  2  2  2  2  2  2  2  4 10
V  2  2  0  0  3  1  1  8  0  6  4  0  0  0  0  0  2 10
W  0  0  0  0  5  0  1  0  0  1  1  0  0  1  3  1  0  0 16
Y  0  1  0  1  9  0  2  3  0  2  3  0  1  1  0  1  0  3  5 12

#
# This matrix indicates how important it is to line up any given
# pair of amino acid structure types. 
#
# B = betaStrand
# C = random coil
# H = helical
# T = beta turn
# X = don't know or don't care 
#

KEY:WT_SMAT		5	
	B   C   H   T   X
B   3
C  -1   2 
H  -3  -1   3
T   0  -1  -1   2
X   0   0   0   0   0


#
# If this matrix is used in sequence alignments, what would be
# the penalty for introducing a gap in the alignment. Increasing
# this number reduces the overall number of gaps in your alignment.

KEY:GAP_PEN 		10

#
# Enter the penalty for each blank in the gap (ie, gap size).
# Should a big gap be penalized more than a small gap? In most
# cases, yes.  However this penalty can be zero or some small
# number if the sequences are roughly the same length. Increase
# this penalty if your alignment contains large unrealistic gaps.

KEY:GAP_PEN_SIZE	2

#
# In the above smiliarity matrix specify the score that would constitute
# a near match between amino acids. This is used for printout purposes.

KEY:NEAR_MATCH		5

#
# Indicate the penalty for inserting a gap in the middle of a
# helix or beta strand structure (if known).
#
# Note that this is a penalty in addition to the regular gap
# penalty as described above.

KEY:MID_GAP_PEN 	30

#
# Indicate the penalty for inserting a gap near the end of a
# helix or beta strand structure.
#
# Typically this value is 3 or 4 times less than the middle of
# structure penalty directly above.

KEY:END_GAP_PEN 	10

#
# The above similarity matrix does not indicate the score of matching
# an amino acid with a blank (eg, a gap in an alignment). Matching a
# blank with an amino acid in a multiple alignment is not necessarily
# a bad thing (particularly gaps before or after a sequence).

KEY:BLANK_MATCH		0