It is recommended that the depth chart be completely filled in, though not required. All position slots that would be used in a game based upon the formations you use should have a player listed. Depending upon the position, the number one player may be the only one to get into the game, but his backups should be set in case of injury. Additionally, the basic depth chart determines who your starters are for the game.
If a player is on your roster but not in the depth chart, they are still available to play. If during the game the simulator needs a player at a position due to injury or fatigue and there are no more players in the depth chart, it will take the next available player at that positions. For instance, if you have no WRs set in your depth chart and the simulator is putting a WR on the field, it will pick one that is available from your roster. Likewise, if you only had one WR in your depth chart and he needs to come out due to fatigue or injury, it will pick an available WR from your roster.