I believe the answer you are looking for would be the second answer choice, "We read Shakespeare's tragedy, <em>Othello</em>, after reading two of his comedies."
This is because when information already speaking of something mentioned, is referred to to clarify a subject, it must be separated by commas. (that's how I remember)
ex: My mother, the queen, is placing a new law down to make everyone have equal rights. ex: Today I went to Ms. Green's, the principal's, office.
