>I
CATCAGTATAAAATGACTAGTAGCTAGATACCACAGATACGATACAACA
>II
TACCACAGATACGATACAACACATCAGTATAAAATGACTAGTAGCAGAC

##</code><code>##

I	2	A	G
I	4	C	T
I	5	A	G
I	7	T	C
II	1	T	C
II	2	A	G
II	3	C	T
II	5	A	C
II	8	G	T
II	10	T	G

##</code><code>##

$F0 = DNA sequence the changes should be made to
$F1 = Letter position that is involved in the change
$F2 = Pre-existing letter
$F3 = Replacement letter

##</code><code>##

#!/usr/bin/env perl
use strict;
use warnings;
use Bio::SeqIO;

#Read and store .FASTA file
my %sequences;
my $seqio = Bio::SeqIO->new(-file => $ARGV[0]);
while(my $seqobj = $seqio->next_seq) {
	my $id  = $seqobj->display_id;
	my $seq = $seqobj->seq;
	$sequences{$id} = $seq;
}

#Variant Input File & Output
my $variants = $ARGV[1];
open IN, '<', $variants or die "$!";
open OUT, '>', 'output.txt' or die "$!";

#Loops
my $iteration = 0;
foreach my $key (sort keys%sequences) {
	$iteration ++;
	my $value = $sequences{$key};
	while (<IN>) {
		my (@F) = split("\t", $_);
		if ($F[0] eq $key) {
			substr($value,0,$F[1]) = $F[3];
		} else {
			last 
		}
	}
	print OUT ">$key\n$value" if $iteration=~1;
	print OUT "\n>$key\n$value" if $iteration>1;
}

##</code><code>##

substr($value,$F[1]-1,1) = $F[3];
use 'next' instead of 'last'
added seek(IN,0,0);