• The first 2 commented lines are incorrect because they attempt to create hashrefs that are based on lists that contain an odd number of elements
• The next 2 assignments are correct, and equal in syntax
• Your code assigning keys(%hash) to %large_key will simply assign the first available key. Why not assign the smallest number to it ? (Need some research to find out this value)
• If keys are numeric (As indicated by your use of ">"), would an ARRAY be more appropriate ?
• You could use the max function in List::Util to replace "sub largest_key".
• Using "sort" to find "max" is overkill. Use max from List::Util.

Thanks for the comments. I have significantly reworked the code, and most of it is working correctly. I can read in the blast results table, correctly assign "Flags" to each row of results. Find the maximum $bitscore for each query. But in the last section, I want to tabulate the number of different "Flags" for each Query, only for those results with $bit_scores within 90% of the top bit score. However, there is an error -> see below. Any help appreciated.

############### read in blast table and parse #######
my $in_blast_tab=$ARGV[0];
open(IN,$in_blast_tab) or die "cannot open $in_blast_tab\n";

my $HoFlg1={}; my $HoFlg2={};
#my $Flag1; my $Flag2;
my %maxBits; my $max_bits=60;

while (my $line=<IN>) {
        chomp $line;
        my ($Query_id,$strand,$Subj_id,$Perc_iden,$align_len,$num_mm,$
+gap,$q_start,$q_end,$s_start,$s_end,$e_value,$bit_score)=split("\t",$
+line); # extra f
ield of strand
        next if ($bit_score <60);
        if ($bit_score > $max_bits){
                $max_bits=$bit_score;
        }
        $maxBits{$Query_id}=$max_bits;
        my ($Flag1, $Flag2 ) = &Flag( $Subj_id, \%proph_prots, \%euk_p
+rots, \%vir_prots );
        $HoFlg1->{$Query_id}->{$Flag1}->{$bit_score}++;
        $HoFlg2->{$Query_id}->{$Flag2}->{$bit_score}++;
        print join("\t",$Query_id,$Subj_id,$bit_score,$Flag1,$Flag2)."
+\n";
}
## Get a Flag for each query/subject 
sub Flag {
    my ( $Subj_id, $proph_prots, $euk_prots, $vir_prots ) = @_;
    return "Proph", "Phage" if exists $$proph_prots{$Subj_id};
    return "Euk",   "Euk"   if exists $$euk_prots{$Subj_id};
    return "Vir",   "Phage" if exists $$vir_prots{$Subj_id};
    return "Bact",  "Bact";
} ## end sub Flag

## now for all query/flag pairs with bit scores that are within 90% of
+ the top_bit for that query
## 
my @flag_list2=("Phage","Euk","Bact");
my $count={};
foreach my $q (keys %{$HoFlg2}){
print "$q\t";
        for my $flag (@flag_list2){
                for my $b (keys %{$HoFlg2->{$flag}}){
                        if ($b > 0.9*$maxBits{$q}){
                                $count->{$flag} += $HoFlg2->{$q}->{$fl
+ag}->{$b};
                        }

                }
        print "$count\t";
        }
print "\n";
}
[download]

sampe input

158256496-stool1_revised_C972998_1_gene3        strand:-        581103
+.GY4MC1_2020      37.93   116     61      2       1       110     1  
+     111     1e-
158256496-stool1_revised_C972998_1_gene3        strand:-        539329
+.FTPG_01302       36.28   113     71      1       1       113     1  
+     112     1e-
158256496-stool1_revised_C972998_1_gene3        strand:-        634956
+.Geoth_2108       37.93   116     61      2       1       110     1  
+     111     1e-
V1.UC9-0_revised_scaffold1508_3_gene71913       strand:+        565641
+.EFOG_02198       57.55   212     55      3       1       212     967
+     1143    9e-
V1.UC9-0_revised_scaffold1508_3_gene71913       strand:+        565640
+.EFMG_02145       57.55   212     55      3       1       212     967
+     1143    9e-
V1.UC9-0_revised_scaffold1508_3_gene71913       strand:+        565639
+.EFYG_01514       57.55   212     55      3       1       212     967
+     1143    9e-
V1.UC9-0_revised_scaffold1508_3_gene71913       strand:+        565638
+.EFCG_01989       57.55   212     55      3       1       212     967
+     1143    9e-
[download]

curent output

158256496-stool1_revised_C972998_1_gene3    HASH(0x2b3c2bd3b000)    HA
+SH(0x2b3c2bd3b000)    HASH(0x2b3c2bd3b000)    
V1.UC9-0_revised_scaffold1508_3_gene71913    HASH(0x2b3c2bd3b000)    H
+ASH(0x2b3c2bd3b000)    HASH(0x2b3c2bd3b000)
[download]

desired output

#query num_Phage num_Euk num_Bact
query_1   5        1       0
query_2   0        6       1
[download]

Could it be the line: print "$count\t";

Should it be print "$count->{$flag}\t";?

Cristoforo (++) is right.

A few more pieces of advice:

• use strict;. If you did, it would have told you that "$count" was not valid.
• Do not pre-pend your called subroutine names with &. There is old fashioned, and there are a few esoteric reasons for using it (Beyond the scope of this note).
• Your "$max_bits" can be initialized to zero - that is probably more understable to the lay reader than having to explain why you start at 60.

             I hope life isn't a big joke, because I don't get it.
                   -SNL

I'm finding it difficult to understand what data structures you are trying to create and subsequently access. I think it would help us to help you if you could provide a small sample of the data you are working with and a description of what you are trying to do with it. Perhaps you could use Data::Dumper to confirm that the data structures you create are what you expected.