When I started the insane journey of seeking publication, I encountered roadblocks right away. Not the agent's genre, poorly written, unlikeable characters, confused plot, etc.
These were all great lessons and I have since been able to solve the majority of those problems in most of my projects.
Now I'm much closer in my journey. I've had times where I was millimeters away from getting representation, but was turned down in the end. I've received enough feedback to learn that I missed my chance due to some of THE LITTLE THINGS.
Those little things can make you or break you. Here a few:
1.) WORD COUNT: I did a post on it
2.) Character motivation, (Even the little ones): If a character does something that doesn't make sense, that could be your death knell.
3.) Typos/ punctuation errors: Agents and publishers are getting less "editorial" every day. The closer a manuscript is ready to be set in type, the better. Also, you want to look like a professional, right?
4.) Wandering away from the story: Stephen King says that editing is "Taking out all the things that are not the story" This is where your betas achieve sainthood, but you must also be vigilant. Don't let side characters hijack the story.
5.) Adverbs and cliches: An agent once said about one of my projects, "The fine line between satire and cliche was a bit indistinct for my taste" Though I was like, "Score! She spotted the satire!" I went back to work on making the manuscript more original. Adding depth to the characters and their motivation really helped with that.
In short, your manuscript must be perfect when you submit it. Not just "pretty good" but perfect. Scared yet? Good, I'd love the company! :)