I can't tell because I don't know what the lines you are dealing with look like. I can only help with overall structure and technique. On the face of it the handling looks OK, but it does depend very much on the content of the first two fields on each line. Can the letter case of the contents be different or can numeric representation be different between the fields for example?

I've applied the changes I suggested in my reply along with a few others to give:

#!/usr/bin/perl
use strict;
use warnings;

sub iterblast {
    # settings
    my $myblast = "blastall -p blastn -m 8";
    my $starterseq =
        "/share/home/tjuh/Thema12-2011/Project/testblast/matchedhits.f
+asta";
    my $blastdb =
        "/share/home/tjuh/Thema12-2011/Project/databases/termite_hindg
+ut/hindgut_db";
    my $outputroot = "/share/home/tjuh/Thema12-2011/Project/testblast/
+test1";
    my $evalue     = 0.01;
    my $wordsize   = 11;
    my $roundlimit = 4;

    # output files:
    my $seq_db_fasta = $outputroot . ".tmp";
    my $seq_db_all   = $outputroot . ".all";
    my $logfile      = $outputroot . ".log";

    # remove existing files
    unlink $seq_db_fasta;
    unlink $seq_db_all;
    unlink $logfile;

    open my $log, '>', $logfile or die "Can't open $logfile: $!\n";
    print $log <<LOGMSG;
Configurations:
Input file       : $starterseq
Output files     : $outputroot\-
Blast Engine     : $myblast
Target database  : $blastdb
E-value cut-off  : $evalue
Blast word size  : $wordsize
Blast round limit: $roundlimit

Runs:
LOGMSG

    # hits hash
    my %hits;
    my %queryids;

    # Start the iterative blastn rounds
    for my $round (1 .. $roundlimit) {
        my $saveblst = $outputroot . ".txt";
        
        unlink $saveblst;

        # print $log info
        my $curtime = localtime();
        my $infile = $round == 1 ? $starterseq : $seq_db_fasta;

        print $log "$curtime - Blast seq: $infile\n";
        print $log "$curtime - Blast db : $blastdb\n";

        # run blast
        system(
            "$myblast -i $infile -d $blastdb -o $saveblst -e $evalue -
+W $wordsize"
            );

        open my $parse, '<', $saveblst or die "Can't open $saveblst: $
+!\n";
        open my $fasta, '>', $seq_db_fasta
            or die "Can't create $seq_db_fasta: $!\n";

        # parse blast output (tab form) and add matched id's in hits h
+ash
        while (defined (my $line = <$parse>)) {
            chomp $line;
            my @fields = split(/\t/, $line);
            ++$queryids{$fields[0]};
            ++$hits{$fields[1]};
        }

        # get header and sequence with 'fastacmd' and write to new fas
+ta file
        my $hitcount;

        while (my $key = each %hits) {
            next if exists $queryids{$key};

            # only write uniq ids to file
            system("fastacmd -d $blastdb -s 'lcl|$key' >> $seq_db_fast
+a");
            $hitcount++;
        }

        close $fasta;
        close $parse;

        if ($hitcount) {
            print $log "Round $round has $hitcount unique hits\n\n";
            next;
        }

        print $log "No hits in Round $round ==> STOP\n";
        last;
    }

    print "Total uniq hits found in all rounds: ", scalar keys %hits, 
+"\n";
    close($log);
}
[download]

Note that I've eliminated system calls to delete files and get the date and replaced them with the equivalent Perl functions! So far as I can see there are no changes in the logic of the code, but to my mind it is somewhat cleaner.

Thank u GrandFather for cleaning up the code a bit, much appreciated

Im adding an example blastoutput ($saveblast) of the first and second round so u can see if im doing it rite

Round 1 blast output

2004112448    2004112448    100.00    738    0    0    1    738    1  
+  738    0.0    1421
2004112448    2004113832    90.44    586    54    2    153    738    1
+6    599    0.0     660
2004112448    2004101124    89.67    368    37    1    361    728    1
+    367    3e-104     379
2004112448    2004105206    83.33    606    101    0    76    681    4
+    609    1e-100     367
2004112448    2004086271    85.95    363    51    0    341    703    8
+3    445    5e-75     281
2004112448    2004120286    80.98    205    39    0    208    412    3
+73    577    1e-19    97.6
2004112448    2004133966    91.43    70    6    0    618    687    261
+    192    9e-18    91.7
2004112448    2004136134    87.84    74    9    0    193    266    345
+    418    5e-13    75.8
2004112448    2004112239    84.34    83    13    0    461    543    10
+1    183    8e-09    61.9
2004112448    2004112239    89.36    47    5    0    366    412    6  
+  52    2e-06    54.0
2004112448    2004097170    85.14    74    11    0    193    266    34
+6    419    3e-08    60.0
2004112448    2004127588    86.15    65    9    0    193    257    346
+    410    1e-07    58.0
2004112448    2004119982    86.15    65    9    0    193    257    346
+    410    1e-07    58.0
2004112448    2004100435    86.15    65    9    0    193    257    55 
+   119    1e-07    58.0
2004112448    2004135256    82.56    86    15    0    184    269    33
+7    422    8e-06    52.0
2004112448    2004121539    81.93    83    15    0    193    275    34
+6    428    5e-04    46.1
2004112448    2004092379    81.93    83    15    0    193    275    34
+6    428    5e-04    46.1
2004099281    2004099281    100.00    844    0    0    1    844    1  
+  844    0.0    1661
2004099281    2004142218    90.13    446    43    1    400    844    1
+    446    1e-149     529
2004099281    2004092058    81.88    458    82    1    376    832    7
+6    533    1e-63     244
2004099281    2004147330    83.64    110    18    0    424    533    5
+41    650    6e-13    75.8
2004099281    2004147330    82.86    70    12    0    230    299    34
+7    416    0.002    44.1
2004099281    2004146484    86.05    86    12    0    685    770    57
+7    662    6e-13    75.8
2004099281    2004146484    85.88    85    12    0    424    508    31
+6    400    2e-12    73.8
2004099281    2004146484    83.10    71    12    0    229    299    12
+1    191    5e-04    46.1
2004099281    2004106112    81.68    131    23    1    694    823    6
+13    743    2e-09    63.9
2004099281    2004147824    85.71    70    10    0    439    508    49
+0    559    4e-08    60.0
2004099281    2004153303    88.64    44    5    0    439    482    619
+    662    1e-04    48.1
2004099281    2004082897    81.52    92    17    0    664    755    10
+    101    1e-04    48.1
2004099281    2004142389    89.47    38    4    0    795    832    169
+    206    0.002    44.1
[download]

round 2 blast output

2004082897    2004082897    100.00    810    0    0    1    810    1  
+  810    0.0    1582
2004082897    2004156684    87.74    261    32    0    3    263    153
+    413    1e-69     264
2004082897    2004149613    80.35    514    99    2    289    801    1
+    513    2e-53     210
2004082897    2004146484    87.91    91    11    0    8    98    554  
+  644    2e-18    93.7
2004082897    2004126309    81.10    164    31    0    8    171    80 
+   243    4e-14    79.8
2004082897    2004106112    85.11    94    14    0    8    101    581 
+   674    6e-13    75.8
2004082897    2004127134    95.45    44    2    0    127    170    745
+    788    9e-12    71.9
2004082897    2004127134    91.18    34    3    0    223    256    844
+    877    0.002    44.1
2004082897    2004117857    79.46    185    38    0    621    805    4
+5    229    9e-12    71.9
2004082897    2004142219    87.69    65    8    0    25    89    904  
+  968    6e-10    65.9
2004082897    2004142219    90.57    53    5    0    136    188    101
+5    1067    6e-10    65.9
2004082897    2004142389    100.00    29    0    0    571    599    60
+1    629    1e-07    58.0
2004082897    2004142389    96.43    28    1    0    143    170    170
+    197    1e-04    48.1
2004082897    2004092058    83.15    89    15    0    13    101    367
+    455    1e-07    58.0
2004082897    2004142218    87.27    55    7    0    10    64    265  
+  319    2e-06    54.0
2004082897    2004117248    96.67    30    1    0    142    171    667
+    696    8e-06    52.0
2004082897    2004125016    96.55    29    1    0    571    599    571
+    599    3e-05    50.1
2004082897    2004125016    96.15    26    1    0    145    170    142
+    167    0.002    44.1
2004082897    2004099281    81.52    92    17    0    10    101    664
+    755    1e-04    48.1
2004082897    2004117249    93.10    29    2    0    571    599    244
+    272    0.008    42.1
2004083856    2004083856    100.00    774    0    0    1    774    1  
+  774    0.0    1534
2004083856    2004128009    87.09    767    84    3    13    764    41
+2    1178    0.0     735
2004083856    2004100486    89.04    365    40    0    408    772    1
+23    487    1e-112     406
2004083856    2004112466    91.47    211    18    0    1    211    598
+    808    3e-73     276
2004083856    2004147543    84.54    194    30    0    346    539    8
+71    1064    2e-34     147
2004083856    2004131474    84.74    190    29    0    346    535    8
+86    1075    2e-34     147
2004083856    2004131474    87.80    41    5    0    61    101    613 
+   653    0.008    42.1
2004083856    2004131474    82.61    69    12    0    138    206    69
+0    758    0.008    42.1
2004083856    2004097313    89.36    94    10    0    679    772    76
+    169    2e-22     107
2004083856    2004149567    90.91    44    4    0    73    116    235 
+   278    5e-07    56.0
[download]

As u can see for example 2004099281 and 2004082897 in the second column, they appear in both rounds, and what im trying to do is to only get the new uniq hits that are to be used in new rounds. I hope i made myself a bit clear, its a bit different to explain.

I started working through your code to eliminate the system calls and modify the file I/O to use string files when I realised that toolic had already identified a major bug with your code. See his second bullet point in Re: Iterative BLAST(N) program. toolic also makes the very strong point that you should write some test code or otherwise validate your results using a test set of data.

It may be that the following self contained sample code provides a test bed for you to determine what you need to know:

#!/usr/bin/perl
use strict;
use warnings;

my @roundData = (<<DATA1, <<DATA2);
2004112448    2004112448    100.00    738    0    0    1    738    1  
+  738    0.0    1421
2004112448    2004113832    90.44    586    54    2    153    738    1
+6    599    0.0     660
2004112448    2004101124    89.67    368    37    1    361    728    1
+    367    3e-104     379
2004112448    2004105206    83.33    606    101    0    76    681    4
+    609    1e-100     367
2004112448    2004086271    85.95    363    51    0    341    703    8
+3    445    5e-75     281
2004112448    2004120286    80.98    205    39    0    208    412    3
+73    577    1e-19    97.6
2004112448    2004133966    91.43    70    6    0    618    687    261
+    192    9e-18    91.7
2004112448    2004136134    87.84    74    9    0    193    266    345
+    418    5e-13    75.8
2004112448    2004112239    84.34    83    13    0    461    543    10
+1    183    8e-09    61.9
2004112448    2004112239    89.36    47    5    0    366    412    6  
+  52    2e-06    54.0
2004112448    2004097170    85.14    74    11    0    193    266    34
+6    419    3e-08    60.0
2004112448    2004127588    86.15    65    9    0    193    257    346
+    410    1e-07    58.0
2004112448    2004119982    86.15    65    9    0    193    257    346
+    410    1e-07    58.0
2004112448    2004100435    86.15    65    9    0    193    257    55 
+   119    1e-07    58.0
2004112448    2004135256    82.56    86    15    0    184    269    33
+7    422    8e-06    52.0
2004112448    2004121539    81.93    83    15    0    193    275    34
+6    428    5e-04    46.1
2004112448    2004092379    81.93    83    15    0    193    275    34
+6    428    5e-04    46.1
2004099281    2004099281    100.00    844    0    0    1    844    1  
+  844    0.0    1661
2004099281    2004142218    90.13    446    43    1    400    844    1
+    446    1e-149     529
2004099281    2004092058    81.88    458    82    1    376    832    7
+6    533    1e-63     244
2004099281    2004147330    83.64    110    18    0    424    533    5
+41    650    6e-13    75.8
2004099281    2004147330    82.86    70    12    0    230    299    34
+7    416    0.002    44.1
2004099281    2004146484    86.05    86    12    0    685    770    57
+7    662    6e-13    75.8
2004099281    2004146484    85.88    85    12    0    424    508    31
+6    400    2e-12    73.8
2004099281    2004146484    83.10    71    12    0    229    299    12
+1    191    5e-04    46.1
2004099281    2004106112    81.68    131    23    1    694    823    6
+13    743    2e-09    63.9
2004099281    2004147824    85.71    70    10    0    439    508    49
+0    559    4e-08    60.0
2004099281    2004153303    88.64    44    5    0    439    482    619
+    662    1e-04    48.1
2004099281    2004082897    81.52    92    17    0    664    755    10
+    101    1e-04    48.1
2004099281    2004142389    89.47    38    4    0    795    832    169
+    206    0.002    44.1
DATA1
2004082897    2004082897    100.00    810    0    0    1    810    1  
+  810    0.0    1582
2004082897    2004156684    87.74    261    32    0    3    263    153
+    413    1e-69     264
2004082897    2004149613    80.35    514    99    2    289    801    1
+    513    2e-53     210
2004082897    2004146484    87.91    91    11    0    8    98    554  
+  644    2e-18    93.7
2004082897    2004126309    81.10    164    31    0    8    171    80 
+   243    4e-14    79.8
2004082897    2004106112    85.11    94    14    0    8    101    581 
+   674    6e-13    75.8
2004082897    2004127134    95.45    44    2    0    127    170    745
+    788    9e-12    71.9
2004082897    2004127134    91.18    34    3    0    223    256    844
+    877    0.002    44.1
2004082897    2004117857    79.46    185    38    0    621    805    4
+5    229    9e-12    71.9
2004082897    2004142219    87.69    65    8    0    25    89    904  
+  968    6e-10    65.9
2004082897    2004142219    90.57    53    5    0    136    188    101
+5    1067    6e-10    65.9
2004082897    2004142389    100.00    29    0    0    571    599    60
+1    629    1e-07    58.0
2004082897    2004142389    96.43    28    1    0    143    170    170
+    197    1e-04    48.1
2004082897    2004092058    83.15    89    15    0    13    101    367
+    455    1e-07    58.0
2004082897    2004142218    87.27    55    7    0    10    64    265  
+  319    2e-06    54.0
2004082897    2004117248    96.67    30    1    0    142    171    667
+    696    8e-06    52.0
2004082897    2004125016    96.55    29    1    0    571    599    571
+    599    3e-05    50.1
2004082897    2004125016    96.15    26    1    0    145    170    142
+    167    0.002    44.1
2004082897    2004099281    81.52    92    17    0    10    101    664
+    755    1e-04    48.1
2004082897    2004117249    93.10    29    2    0    571    599    244
+    272    0.008    42.1
2004083856    2004083856    100.00    774    0    0    1    774    1  
+  774    0.0    1534
2004083856    2004128009    87.09    767    84    3    13    764    41
+2    1178    0.0     735
2004083856    2004100486    89.04    365    40    0    408    772    1
+23    487    1e-112     406
2004083856    2004112466    91.47    211    18    0    1    211    598
+    808    3e-73     276
2004083856    2004147543    84.54    194    30    0    346    539    8
+71    1064    2e-34     147
2004083856    2004131474    84.74    190    29    0    346    535    8
+86    1075    2e-34     147
2004083856    2004131474    87.80    41    5    0    61    101    613 
+   653    0.008    42.1
2004083856    2004131474    82.61    69    12    0    138    206    69
+0    758    0.008    42.1
2004083856    2004097313    89.36    94    10    0    679    772    76
+    169    2e-22     107
2004083856    2004149567    90.91    44    4    0    73    116    235 
+   278    5e-07    56.0
DATA2

iterblast();

sub iterblast {
    # settings
    my $myblast = "blastall -p blastn -m 8";
    my $blastdb = 'foo';
    my $evalue     = 0.01;
    my $wordsize   = 11;
    my $roundlimit = 4;

    print <<LOGMSG;
Configurations:
Output files     : xxx\-
Blast Engine     : $myblast
Target database  : $blastdb
E-value cut-off  : $evalue
Blast word size  : $wordsize
Blast round limit: $roundlimit

Runs:
LOGMSG

    # hits hash
    my %hits;
    my %queryids;
    my @fastaDbs;

    # Start the iterative blastn rounds
    for my $round (1 .. @roundData) {
        open my $parse, '<', \$roundData[$round - 1];

        push @fastaDbs, '';
        open my $fasta, '>', \$fastaDbs[-1];

        # parse blast output (tab form) and add matched id's in hits h
+ash
        while (defined (my $line = <$parse>)) {
            chomp $line;
            my @fields = split(/\t/, $line);
            ++$queryids{$fields[0]};
            ++$hits{$fields[1]};
        }

        # get header and sequence with 'fastacmd' and write to new fas
+ta file
        my $hitcount;

        for my $hit (keys %hits) {
            next if exists $queryids{$hit};

            # only write uniq ids to file
            print $fasta "fastacmd -d $blastdb -s 'lcl|$hit'\n";
            $hitcount++;
        }

        close $fasta;
        close $parse;

        if ($hitcount) {
            print "Round $round has $hitcount unique hits\n\n";
            next;
        }

        print "No hits in Round $round ==> STOP\n";
        last;
    }

    print '-' x 20, "\n$_\n" for @fastaDbs;
    print "Total uniq hits found in all rounds: ", scalar keys %hits, 
+"\n";
}
[download]

The major interesting technique here (used in various ways in several places) is using a variable as a file. That avoids writing or reading any actual files at all, but allows most of the interesting processing code to remain intact.

True laziness is hard work