Okay, here's another mess of observations made the second time through:
Yes, it's possible to get to Genesis without the Snake and any of the ammo.
There's that 5 missile box in Grasslands. This is another item that could be avoided and taken after Genesis, but you could simply replace it with a health box (and then replace that health box up high with the 5 missiles or something); there's not much to it, but it'd be nice to reward someone for that trick for more than one level.
I'm not sure if it was disabling the terminal on the far left of Grasslands, or if it was a terminal that is supposed to be out of reach (if not for a damage hop off a bat), but you can disable the fan that pushes the player away from the Explosive (yes, it's get-able).
Regarding the end of sky Genesis, some kind of hint would help, but it can be more subtle than a sign. That said, I would lean towards not penalizing a player so much if they don't get it (the Bubbler is a pretty big dropoff from the Blade, especially after de-leveling).
In the Sand Zone, I'm going to agree about that one chest completely surrounded by solid blocks. It's not enough to say there's an item there; you also have to give some kind of hint of how to reach it (the same goes for one of those boxes in the Labyrinth). Also, the hint about Mimigas and Red Flowers living in peace is a bit misleading (I was trying to get them to both stop barking, not have them be the only two barkers); if anything, it would make more sense to say something about the Gaudi ending their grudge, or something.
The boss here is neat, but most of the room doesn't come into play. *shrugs*
In the Labyrinth, in addition to being able to skip Monster X (yeah, I now see the column of spikes is avoidable, and yes the 3 HP run is impossible if you skip Monster X), it's also possible to skip the memory chip for the teleporter; there's nothing that seems to force you to get it.
That big critter makes for an interesting Balfrog replacement; I'm actually impressed you were able to make it "work" (since Balfrog fights typically didn't function properly in hacks).
I also realized I "missed" a Super Missile upgrade. If that upgrade is going to be hidden, I'm going to recommend more strongly that you have a better un-missable weapon available (like the machinegun). Or maybe you award other weapons and then allow for a barter system for upgrades (trade two crappy weapons to get one decent weapon); I'll see if I can come up with some ideas for that, but it'll be kind of tricky if you're going to have Blade (a moderate/decent weapon) available so early as a secret, and if you have your heart set on certain weapons being availalbe at certain times, it's probably just not going to work.
In the Revolutionariam, there's a BuyoBuyo Base before the 'teer fight. If you don't kill it, you can use it to harvest weapon energy and health in case you take damage. This is almost essential for a Bubbler + normal Missile player, but you probably want to block that area off during the boss fight.
I did at least get my Missile count up to 20 on this playthrough. So my chestlist has me missing the Missile upgrade, 13 HP, and Remote Control. But other items I've been able to have that weren't on that list are: Snake (delayed gratification), Explosive (turned off the offending fans), Lipstick (o_O), and Critter Juice (a few spikes never killed anyo... err, nevermind).
I'm pretty sure that's it.