The tr/// changes non-zero bytes to x. The while loop then uses index to search through the difference string for the x bytes and reports their index (0 based position).

I think it might be simpler to use a global regex match for non-nulls and pos. You can just match them for 1-based counting or put them in a look-ahead for 0-based counting. Note that I added another difference in the third characters to check that it was working as I had hoped.

use strict;
use warnings;

my $one  = "AGCTGATCGAGCTAGTACCCTAGCTC";
my $two  = "AGATGATCGAGCTAGTACCCTATCTC";
# Diffs here  ^                   ^

my $diff = $one ^ $two;

my @posns = ();

push @posns, pos $diff while $diff =~ m{[^\0]}g;
print qq{Differences, 1-based counting, at - @posns\n};

@posns = ();

push @posns, pos $diff while $diff =~ m{(?=[^\0])}g;
print qq{Differences, 0-based counting, at - @posns\n};
[download]

The output.

Differences, 1-based counting, at - 3 23
Differences, 0-based counting, at - 2 22
[download]

I hope this is of interest.

Cheers,

JohnGG

sxoght: #query  hit     score   probability     qstart  qend    qorien
+tation    tstart  tend    matches mismatches      gapOpening      gap
+s
@SNPSTER4_104_308EFAAXX:1:1:1694:128
GGGATAAGAGAGGTGCATGTTGGTATTTAAGGTAGT
1 alignment(s) -- reports limited to 10 alignment(s)

sxoght: SNPSTER4_104_308EFAAXX:1:1:1694:128     gi|122939163|ref|NM_00
+0165.3|   -10     1.000000        1       36      +       1595    163
+0    35      1   00
Score = -10, P(A|R) = 1.000000
Query:          1 GGGATAAGAGAGGTGCATGTTGGTATTTAAGGTAGT 36
                  |||||||||||||||||||||||||||||| |||||
Sbjct:       1595 GGGATAAGAGAGGTGCATGTTGGTATTTAAAGTAGT 1630

@SNPSTER4_104_308EFAAXX:1:1:1608:94
GCAGTTTTAAGTTATTAGTTTTTAAAATCAGTACTT
14 alignment(s) -- reports limited to 10 alignment(s)

sxoght: SNPSTER4_104_308EFAAXX:1:1:1608:94      gi|113412254|ref|XR_01
+8775.1|   0       0.090884        1       36      +       1578    161
+3    36      0   00
Score = 0, P(A|R) = 0.090884
Query:          1 GCAGTTTTAAGTTATTAGTTTTTAAAATCAGTACTT 36
                  ||| |||||||||||||||||||||||||||||| |
Sbjct:       1578 GCATTTTTAAGTTATTAGTTTTTAAAATCAGTACGT 1613
[download]

>gi|122939163|ref|NM_000165.3|    1595    1630    SNPSTER4_104_308EFAA
+XX:1:1:1694:128    1    36    36    -10    1    1.000000    35    
mismatch : 1625.GA        
>gi|113412254|ref|XR_018775.1|    1578    1613    SNPSTER4_104_308EFAA
+XX:1:1:1608:94    1    36    36    0    1    0.090884    36    
mismatch : 1581.GT 1612.TG
[download]

#!/usr/bin/perl
open(FILE,"align.input") or die "can not open file";
while($var=<FILE>){
    $str1=();$str2=();
    if($var=~/^sxoght:/){
        @ar=split(/\s+/,$var);
    print ">$ar[2]\t$ar[8]\t$ar[9]\t$ar[1]\t$ar[5]\t$ar[10]\t$ar[6]\t$
+ar[3]\t$ar[4]\t$ar[11]\n";
    }
    if($var=~/^Query:/){
        $str1=$var;
        $str1=~s/^Query:\s+//g;
        $str1=~s/\d+\s+//g;
        $str1=~s/\s+//g;
    }
    if($var=~/^Sbjct:/){
        $str2=$var;
        $str2=~s/^Sbjct:\s+//g;
        $str2=~s/\d+\s+//g;
        $str2=~s/\s+//g;
    }
    for($i=0;$i<=length($str1);$i++)
    {
    if(substr($str1,$i,1) ne substr($str2,$i,1)){
        print substr($str1,$i,1);
        print substr($str2,$i,1);
        print "$i\n";        
    }
    }
}
[download]

You should only print the output if $str1 and $str2 are both set, and then, clear them so you don't print them again.

The next is very close to your code, but kind of works. You only need to format the output as desired (I don't know where the large numbers come from):

#!/usr/bin/perl
open(FILE,"align.input") or die "can not open file: $!";
while($var=<FILE>){
    if($var=~/^sxoght:/){
        ($str1,$str2)=();
        @ar=split(/\s+/,$var);
        print ">$ar[2]\t$ar[8]\t$ar[9]\t$ar[1]\t$ar[5]\t$ar[10]\t$ar[6
+]\t$ar[3]\t$ar[4]\t$ar[11]\n";
    }
    if($var=~/^Query:/){
        $str1=$var;
        $str1=~s/^Query:\s+//g;
        $str1=~s/\d+\s+//g;
        $str1=~s/\s+//g;
    }
    if($var=~/^Sbjct:/){
        $str2=$var;
        $str2=~s/^Sbjct:\s+//g;
        $str2=~s/\d+\s+//g;
        $str2=~s/\s+//g;
    }
    if(defined $str1 and defined $str2) {
        for($i=0;$i<=length($str1);$i++)
        {
            if(substr($str1,$i,1) ne substr($str2,$i,1)){
                # this is not in the desired format, yet
                print substr($str1,$i,1);
                print substr($str2,$i,1);
                print "$i\n";
            }
        }
        ($str1,$str2)=();
    }
}
[download]

For the format, I propose to store the results in an array, and print "mismatch:" only if the array isn't empty at the end.

        my @mismatch;
        for($i=0;$i<=length($str1);$i++)
        {
            if(substr($str1,$i,1) ne substr($str2,$i,1)){
                push @mismatch, "$i." . substr($str1,$i,1) . substr($s
+tr2,$i,1);
            }
        }
        if(@mismatch) {
            print "mismatch: @mismatch\n";
        }
[download]

After that modification, the output I get for this file is

>hit    tstart    tend    #query    qstart    matches    qend    score
+    probability    mismatches
>gi|122939163|ref|NM_000165.3|    1595    1630    SNPSTER4_104_308EFAA
+XX:1:1:1694:128    1    35    36    -10    1.000000    1
mismatch: 30.GA
>gi|113412254|ref|XR_018775.1|    1578    1613    SNPSTER4_104_308EFAA
+XX:1:1:1608:94    1    36    36    0    0.090884    0
mismatch: 3.GT 34.TG
[download]

p.s. There's a possible speed improvement if you XOR (^) the two strings, you'll get a string of null bytes for where they are the same and non null where they are not:

        my $xor = $str1 ^ $str2;
        while($xor =~ /[^\0]/g) {
            my $i = pos($xor) - 1;  # or: $-[0]
            push @mismatch, "$i." . substr($str1,$i,1) . substr($str2,
+$i,1);
        }
[download]

hi bart, thank u sooo much for the help.. now i know where all i went wrong. will try to avoid such mistakes next time. i am learning a lot from perlmonks.org. thanks again :)

but u did not define here @var and @ar