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    Pre-release 
Amame Ritsuko and Kagami will all suffer Chuck Cunningham Syndrome
I feel like it’s bound to happen. Especially in the case of Kagami since his English VA passed away (like Mayumi)
  • Averted big time with Amame, who becomes a major character in Nirvana Initiative, to the point of being the game's final Somnium, with the most Mental Locks of any character across both games.
  • Kagami could also just be recast
    • This is indeed what ended up happening, with a honorable mention in Kagami's profile honoring his previous voice actor.

The sequel will involve an Evil Knockoff of Aiba
Because that'd be a logical escalation, and will lead to new action scenes as Aiba and her counterpart try to outwit each other. My guess is that in a flip of Saito, Mizuki's nemesis is someone she knows from the start, but is unable to stop because his AI can hack Aiba and threaten to blow her and Mizuki up - the case is discovering his motives and how he got his hands on an AI.
  • Jossed.

The sequel will explain how Mizuki lost her eye, either as a part of a flashback and/or The Reveal.
It could be related to the main case, or it was intentionally removed so that Mizuki could use Aiba.
  • Semi-confirmed; she loses it prior to a Time Skip.

Where the other main characters could be by the time of nirvanA Initiative.
Supposing that it’s been at least 12 years since the first game, so that Mizuki would be old enough to join ABIS. This also assumes that the sequel will follow the Resolution route from the original game.

  • Date: He’d have settled down with Hitomi and helped to raise Mizuki, while also continuing to work at ABIS. He has either retired or is working on another case by the time the game starts, if he’s going to absent from the sequel.
    • Whoof, jossed. He's been missing for six years while Mizuki grows up and becomes an ABIS agent.
  • Hitomi: Settled down with Date, and was a loving mother to Mizuki. In the present day, she would give Mizuki some advice and there could be sweet moments between the two.
    • Semi-jossed(?). She has way less screentime than in the first game, and doesn't interact with Mizuki for much of it. Though she and Date show hints of being able to work things out.
  • Boss: Likely still working at ABIS, though now she’s Mizuki’s boss.
    • Confirmed.
  • Pewter: May or may not have been released after serving time for his involvement in the second Cyclops Killer case. If he has, then he’s back at ABIS, this time helping Mizuki with the Half Body serial murder case.
    • Confirmed.
  • Mayumi: She may have passed away by the start of the game, as she was already suffering from dementia.
  • Ota: He’s either a successful author or taken over and reopened Matsushita Diner to honor his parents and all they’ve done for him. May or may not have married Iris.
    • Confirmed for taking over the diner. Jossed for marrying Iris (he wishes though).
  • Iris: She’s retired from her idol career, and has likely gotten a different job. Depending on if she married Ota or not, she could be helping him run the diner. She and Mizuki would share a closer bond due to them now being adoptive sisters.
    • Jossed. Still an idol.
  • Moma: Still the head of the Kumakura clan, thought it’s not quite certain that Mizuki will go to them for help.
    • Confirmed. And yes, he does help her out.
  • So: With his political career behind him, he might be spending whatever time he has left in relative peace, supposing he’s still alive by then.
    • Confirmed.

The sequel will take place after the Mizuki ending
On the cover of the box, we can see what looks to be Saito's original body, but no sight of Date's body. Since Saito's body was destroyed after the events of the Resolution Route, this probably means Date's still in Saito's body. And since this game is going to have a lot of focus on Mizuki, it might take place after her route. However due to alternate universes converging on one another, characters like Iris and Saito, despite being dead in this timeline, will still appear somehow.

nirvanA Initiative takes place after the the Golden Ending
While the latest trailer and the released box art do suggest that it takes place after one of the many alternative endings where Date doesn't get his original body back and remains in Saito's, this could simply be a case of the trailer doing this to cover up the big twist of the first game. This can be further corroborated by the fact that, while we do see Date in the trailer, he doesn't say a word in it, likely to cover up how Date's (or more accurately, Saito's) voice actor isn't making a re-appearance.
  • Semi-Confirmed. The game does take place after the Golden Ending and it does feature an option that prevents spoilers for the previous game. However, the actual reason Date looks like Saito is that he decided to wear a mask resembling his face for the sake of undercover work and because it's more popular with the ladies.

nirvanA Initiative will reveal that the Ai games and the Zero Escape games are in the same canon
Multiple timelines are a major part of the "Zero Escape" franchise, with each installment having them be a major plotpoint that allows the protagonists to remember things from one timeline to be used in another one. Ai:Somnium Files has this as a plotpoint too that allows Date to know things that he learned from a different timeline. But unlike the ZE games, it never goes into detail how he is able to remember those things and he is completely baffled by it. Now, in the trailer for nirvanA Initiative, a model that can clearly be seen as the spacesuits from Virtues Last Reward appears for a brief moment. On one hand, it could just be reused assets/a fun reference to VLR, but on the other hand it could be a hint leading to a reveal that they both are the same continuity.
  • Semi-Jossed. Zero Escape is explicitly said to be a popular videogame series in this universe, however, at the same time, they could be some kind of strange true-crime documentary games, given how the dates line up in ZE and Ai respectively

The motif of nirvanA Initiative will be the Right Brain-Left Brain divide
The game's story is going to focus on a series of serial killings where the victim's body is left cut in half. Additionally, the game's box and logo has kind of a "two halves" theme going on, with the logo and the stuff in the background being different colors on both sides. Also, we see what looks like two Aibas on both sides. Since Uchikoshi likes tackling pseudoscientific topics in his games, it's possible he's going with the right brain/left brain divide, and this will somehow play into the psync minigames.
  • Pretty on point it turns out, except for the two-Aibas theory and the minigames being half-themed.

    Post-release (SPOILERS) 

The game after this(?) will introduce our third AI
nirvanA Initiative namedrops a third AI by the name of Marco, Bibi's new partner. The producer has gone on to confirm them as non-binary in a twitter thread, and there's an unused model for them in the game's files. A sequel could expand on their character further and even give them a dynamic with the rest of ABIS, including their two older sisters Mizuki and Tama.

Hitomi is secretly just as kinky as Date is
Someone that is Date's soulmate would either have to match or compliment his extreme horniness. It'd be hilarious/sweet/disturbing if Hitomi, alongside her already established kindness and maturity, has a kinky side to her that only he's seen. Also the potential reactions of horror from Mizuki and Ryuki would be hilarious.

Bibi is Shoko's biological daughter
Shoko had a daughter at a young age and adopted her out. Maybe Shoko had an unplanned pregnancy and a Shotgun Wedding with Renju. Maybe she participated in the genetic experiments, and Renju may or may not have been involved. In any case, the daughter was subject to experiments and cloned, and Shoko and Renju adopted the clone later on. Thus, Mizuki Date is biologically related to Shoko.

Amame and Shoma's desire for revenge is In the Blood from their mom
Amame and Shoma both decided to kill Tearer independently. Komeji doesn't seem like the vengeful type, so maybe their mom is a vengeful person and they got it from her.

The third game will have a subtitle with the initials "SAN"
Since the second game's subtitle, nirvanA Initiative, has the initials "NI" (2), the third game will have the initials "SAN" (3).
  • Sand; Cassandra; Santa; Chrysanthemum...

Amame will get out of jail thanks to Boss's connections
Assuming the third game takes place after the main route, Mizuki will ask Boss to pull some strings to get Amame out of jail. If Boss could get Pewter out within three months (the length of time between the resolution and epilogue of the first game), she could do the same for Amame.

The next game will involve a Frankenstein's Monster as the inspiration of the next course of killings.
The first game specifically included eyes, and this game involved victims sliced down the middle. The next killer will start slicing up victims starting with their head, then working their way down. Not even people suspected of being the killer are safe from being victims, whether they have a specific part the killer needs, or they were in the wrong place and time.

Amame's prison sentence will be a plot point
Several major characters have connections with her, which for a potential Big Bad could be used as leverage over the heroes. They could have other inmates making attempts on her life and use that to blackmail her friends and family, or maybe they're someone in the law system and tampering with her sentence. By the end though, she fulfills her sentence and can reunite with everyone.

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