Start by trimming your input data in half until you find a minimal set of input that causes the issue.

If you don't see a bad entry in the input remaining, then sprinkle some print statements through your code, and see where and when that undef is appearing.

PS: If you rewrite

for($i=0; $i<=$#split_aligns_non_barrel; $i++)
{
$separate_hit_non_barrel=$split_aligns_non_barrel[$i];
...
[download]

as

for my $separate_hit_non_barrel (@split_aligns_non_barrel)
{
...
[download]

Then that would simplify your code a fair bit and make it easier to read. Fewer pseudo-global variables and unnecessary worrying about indexes is good too.