@andrewjs18, the error is clear, the challenge can’t be accessed to verify your domain.
Put a simple test file in /path/to/document/root/for/sub.mysite.com/.well-known/acme-challenge/testfile and try to access it using your web browser http://sub.mysite.com/.well-known/acme-challenge/testfile. If you get the content of testfile all is ok, if you receive a 404 Not found something is wrong in your conf.
Also, re-check that you wrote the correct webroot-path for your sub.mysite.com domain when you executed the letsencrypt-auto command.
Cheers,
sahsanu