Another possibility is that one of Twilight's own insecurities or fears takes on a form and becomes the main antagonist. It drives her to betray her friends as it convinces her that the Mane 6 are not as good as she once thought.
Well, it could be that Twilight discovers some sort of dark secret about the Mane 6 and decides to betray them for what she believes is the greater good. Maybe she's been influenced by some external force.
It could be an outsider who manipulates Twilight's friends. This character might be envious of Twilight's popularity or power in the magical realm. They trick Twilight's friends into believing false things about her, making the friends turn against her.
It could be Ares. He might see the Twilight vampires as a new source of power or chaos. He manipulates Percy into believing that the Twilight characters are evil and need to be stopped. So Ares becomes the main antagonist as he's the one driving Percy to betray the Twilight group.
It could be a dark magic user who doesn't want the human - turned - Twilight to succeed. Maybe this antagonist sees the new Twilight as a threat to their own power or plans in the pony world.
Her friends like Pinkie Pie, Rainbow Dash, Applejack, Rarity and Fluttershy are likely to be main characters. Pinkie Pie might bring her usual cheer and parties to celebrate Twilight's new form. Rainbow Dash could be both impressed and a bit competitive, trying to keep up with Twilight's new abilities in her own way. Applejack would offer her down - to - earth advice and support. Rarity might be excited to create new outfits for Twilight as an alicorn. And Fluttershy would be there with her gentle nature, providing a calming presence.
After the initial shock and sadness, Twilight often becomes determined. She decides that she will not let the betrayal break her. In a story where she is abandoned in a dangerous realm, she uses her magic and wits to find her way back and also to expose those who betrayed her, showing that she is stronger than they thought.
Obviously, Twilight Sparkle is a main character. She's known for her intelligence and magic skills in the MLP world. And then there's Scootaloo. She's a young and energetic pony. In this fanfic, their interactions will drive the story. Twilight as the adopter will be responsible for Scootaloo, and Scootaloo will bring new experiences to Twilight's life.
In some fanfictions, Twilight might be betrayed by a new pony in town. This new pony befriends Twilight, gains her trust, and then uses that trust to get access to the library's restricted section. Once inside, they steal a powerful magical artifact, and Twilight is left to take the blame for the security breach, feeling deeply betrayed.
Well, it could be that there is no traditional antagonist. Sam's betrayal could be the main source of conflict, and she could be seen as the antagonist in Danny's eyes for that time being. But if there has to be another force, it could be the entity that influenced Sam to betray Danny in the first place. For example, a powerful and manipulative ghost that has its own agenda and uses Sam as a pawn.
The main characters are Twilight Sparkle, Rainbow Dash, Pinkie Pie, Rarity, Fluttershy, and Applejack, as it's about their fusion. They are the core of the Mane 6 in the My Little Pony universe and so are central to any fanfic involving their fusion.