In general, messages like "Can't call method "location" on an undefined value ..." mean that you are trying to call a method on an object that doesn't exist (either you haven't created it with its constructor or the object has been destroyed). In this case, it appears that the code that is trying to call a method is in one of the Bio::SeqIO modules. That doesn't rule your code out entirely, but it does make it harder to track down the problem. You might check the docs for that series of modules to see if there are any hints there.