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United debutant goalkeeper Andre Onana survived a stoppage-time penalty check after clattering into Sasa Kalajdzic while failing to claim the ball, with O'Neil booked by referee Simon Hooper for protesting against the inactivity of VAR Michael Salisbury.
Despite registering 23 shots on goal - the most by an away side at Old Trafford since Chelsea in 2005 - Raphael Varane's winner 14 minutes from time condemned Wolves to an opening-night defeat.
Speaking in his post-match press conference, O'Neil said he had received an apology from Jon Moss, manager of the Premier League's elite group of officials.
"We have just spoken to Jon Moss and fair play to him for coming straight out and apologising and saying it was a blatant penalty and should have been given," he said.
"I spent the afternoon with him [on Monday], gave up a lot of my day and preparation around trying to understand the new guidelines and trying not to get myself booked on my first game with the new guidelines, which I failed in.
"Fair play to Jon for saying it was a clear and obvious error and he cannot believe that the on-field ref did not give it, cannot believe that VAR didn't intervene.
"It probably made me feel worse actually because once you know you are right you feel worse about leaving with nothing."
In April, Salisbury was dropped for a round of matches following his errors in Brighton's controversial defeat at Tottenham.
Referee Hooper, VAR Sailsbury and assistant VAR Richard West have been dropped for next weekend with the officials not selected for a Premier League game in the second round of fixtures.
Onana: It wasn't a penalty | O'Neil: Refs influenced by Old Trafford
For his part, Onana told Sky Sports that he did not feel a penalty should have been awarded.
"It was just contact between two big guys," said the Manchester United goalkeeper. "I was very calm and confident it would not be a penalty."
But a baffled O'Neil countered: "I feel if you go for the ball and miss it, and clatter into an opposing player that hard, I just don't see how it's not a foul."
The former Bournemouth boss also suggested match officials may have been swayed by the Old Trafford crowd.
"I don't think the officials are trying to favour one side," he said. "I just think it's human nature. If there's a decision you're not sure about, everyone would be impacted by the Old Trafford crowd and the fact it's Manchester United."
'It was a penalty' - Neville & Carney
There was near consensus in the Monday Night Football studio that Wolves should have been awarded a penalty.
"The reason I thought it was a penalty was because Onana wasn't anywhere near the ball," Sky Sports pundit Gary Neville said. "He was always under it, was never getting there, and could have made a decision before he jumped.
"I always used to say if my goalkeeper was coming out they should clatter the lot, he's done that, so in some ways, he has done what I'd want my goalkeeper to do, but the problem is he's nowhere near the ball.
"He's decided to disturb as much as he can and put the attacking player off, and it's a penalty because he wasn't close enough to the ball. If he arrived at the same time and he just missed the ball, it would have been a different story, but I think he knows what he's doing."
Fellow Sky Sports pundit Karen Carney added: "I think he's [O'Neil] right to feel aggrieved. Onana comes out committed, I think he takes his eye off it at the last minute, but he takes out the player. He just clatters him... it's a penalty, no?"
Jamie Carragher, however, did not believe there was enough evidence to overturn the on-field referee's decision not to award the penalty in the first instance.
"I wasn't sure when I initially saw it," he said.
"When it hadn't been given, I didn't think there was any way in the world it would have been overturned. Once the referee hadn't given it, I didn't think it was a big enough mistake for them to overturn."
Gallagher: Salisbury has pushed threshold to 'absolute limit'
Former Premier League referee Dermot Gallagher speaking to Sky Sports News:
"When you look at the images, I feel the referee on the field should get it. If you see where Simon Hooper is stood and how far Andre Onana comes, he comes a long way and Simon can see it all the way through. Onana crashes into the player and it just has to be given as a penalty. It's got to be a foul.
"I think the VAR should have overturned it. But I wonder if referees are being told there's a higher threshold and nobody really knows where that threshold is, because one person's tolerance level is slightly different from somebody else's. I think the VAR Michael Salisbury has pushed it to the absolute limit. It should be a penalty on the field without a doubt and I thought the VAR should have intervened.
"Since Howard Webb came in to PGMOL we've seen a much more open, transparent dialogue with coaches, managers, teams, and the media. Everybody's welcomed that and embraced it.
"I think the fact they've come out and acknowledged it straight away, that's all you can you do. I know Wolves can turn around and say 'that doesn't do us any good', but it does because at least it acknowledges there was a mistake. Once you acknowledge that mistake, you can learn from it. You can ask the two officials how they got into that position and how they can make sure it doesn't happen again."