Hi amitgsir

Not wanting to assume what your criteria is for UMIs that comprise a cluster; could you expand on what you mean by:

2. Column 3 must also be similar, i.e. in each cluster lines with similar UMI allowing 2 mismatch will be clustered together

We need a bit more to go on:

1. What do the [up to 3] UMIs need to have in common to be considered similar?
2. Why is TCACGGTG in the first cluster instead of TCAAAATG?

If it's all the same and it doesn't matter what the UMIs are as long as you have them in sets of 3s; you could try this:

#!perl -slw
use strict;

my ($chromosomes,$DELIMITER) = (undef,'CLUSTER');
while ( <DATA> ) {
    s/\R//g; # remove line breaks;
    my $record = [split /\s+/];
    push @{$chromosomes->{$record->[0]}->{$record->[2]}},[$record->[1]
+,$record->[3]];
}

foreach my $chrM (sort keys %{$chromosomes}) {
    my $cnt = 0; # used to print delimiter

    foreach my $UMI (sort {$a cmp $b} keys %{$chromosomes->{$chrM}}) {
    print $DELIMITER unless $cnt++ % 3;
    print "$chrM\t$_->[0]\t$UMI\t$_->[1]" 
        foreach (sort {$a->[0] <=> $b->[0] or
                   $a->[1] <=> $b->[1]} @{$chromosomes->{$chrM}->{$UMI
+}});

    }
}
__DATA__
chrM:307    0    AGCGGGGA    129
chrM:307    0    AGCGGGGA    130
chrM:307    0    AGCGGGGA    129
chrM:308    0    AGCGGGGA    129
chrM:308    0    AGCGGGGA    130
chrM:308    0    AGCGGGGA    129
chrM:309    0    AGCGGGGA    129
chrM:309    0    AGCGGGGA    130
chrM:309    0    AGCGGGGA    129
chrM:307    0    TCAAAATG    130
chrM:308    0    TCAAAATG    130
chrM:309    0    TCAAAATG    130
chrM:307    0    TCACGGTG    130
chrM:308    0    TCACGGTG    130
chrM:309    0    TCACGGTG    130
chrM:307    0    TCAGCCTG    129
chrM:308    0    TCAGCCTG    129
chrM:309    0    TCAGCCTG    129
chrM:307    0    TCAGGGAG    130
chrM:308    0    TCAGGGAG    130
chrM:309    0    TCAGGGAG    130
chrM:307    1    TCAGGGTG    106
chrM:307    2    TCAGGGTG    130
chrM:307    2    TCAGGGTG    129
chrM:308    1    TCAGGGTG    106
chrM:308    2    TCAGGGTG    130
chrM:308    2    TCAGGGTG    129
chrM:309    1    TCAGGGTG    106
chrM:309    2    TCAGGGTG    130
chrM:309    2    TCAGGGTG    129
[download]

Output

C:\code\perlmonks>perl pm_1198131.pl
CLUSTER
chrM:307        0       AGCGGGGA        129
chrM:307        0       AGCGGGGA        129
chrM:307        0       AGCGGGGA        130
chrM:307        0       TCAAAATG        130
chrM:307        0       TCACGGTG        130
CLUSTER
chrM:307        0       TCAGCCTG        129
chrM:307        0       TCAGGGAG        130
chrM:307        1       TCAGGGTG        106
chrM:307        2       TCAGGGTG        129
chrM:307        2       TCAGGGTG        130
CLUSTER
chrM:308        0       AGCGGGGA        129
chrM:308        0       AGCGGGGA        129
chrM:308        0       AGCGGGGA        130
chrM:308        0       TCAAAATG        130
chrM:308        0       TCACGGTG        130
CLUSTER
chrM:308        0       TCAGCCTG        129
chrM:308        0       TCAGGGAG        130
chrM:308        1       TCAGGGTG        106
chrM:308        2       TCAGGGTG        129
chrM:308        2       TCAGGGTG        130
CLUSTER
chrM:309        0       AGCGGGGA        129
chrM:309        0       AGCGGGGA        129
chrM:309        0       AGCGGGGA        130
chrM:309        0       TCAAAATG        130
chrM:309        0       TCACGGTG        130
CLUSTER
chrM:309        0       TCAGCCTG        129
chrM:309        0       TCAGGGAG        130
chrM:309        1       TCAGGGTG        106
chrM:309        2       TCAGGGTG        129
chrM:309        2       TCAGGGTG        130
[download]

Cheers
Shadowsong