While I'm not trying to speedrun the Fresh File category, I've been wanting to do a Fresh File clear on the 1st run for a while now, and I've been wondering about 1) what is actually available to you on the first run in terms of what appears within the run itself, and 2) what would be some best practices in terms of a build?

Probably my most successful build was one with Dionysus on Attack, Ares on Special, Duo Boon which buffed Hangover rate, and Divine Dash (I think I also had True Shot, but that was pretty much nothing compared to whatever else I had). Could've stood to have Impending Doom, and I really didn't get any Poms that run, but I made it to Lernie before beefing it. The aim I had with that build was to be able to play as defensively as possible, getting in enough hits to put on decent Hangover and then proc Doom, basically trying to get in most of my damage offscreen while I divided my focus. Could've stood to use Cast more, but I had barely any Poms at all let alone any Poms on it, and the 10% Crit chance wasn't showing dividends.

I'd never actually seen how any of the quick kills or wombo combos worked for the Sword until looking at one of the FAQs, so I'd probably stand to try and incorporate those, if less for the speedgame and more so I can just get enemies over with without risking getting hit.

I know for certain Hammers don't appear until Run 3, and the only gods I've seen in Run 1 are Athena (naturally), Artemis, Ares, and Dionysus. No Survival rooms in Tartarus, though also no Story rooms either from what I've seen; no Chaos Gates either, which I think is to be expected. I'm also wondering if maybe there's some behind-the-scenes work in terms of enemy stats or number, but that could just be me being antsy about these runs.

So you nailed most of the differences. Calls are unlocked run 6, Hammers are unlocked run 4, story rooms require first seeing each of their respective biome bosses (and thus are not available first run). Chaos and Hermes are also not available and you cannot see wells of charon except at the midbiome rooms. As you mentioned, there are only 4 gods available (athena, artemis, ares and dionysus)

The numeric values of the enemies are identical, you are just much weaker without any upgrades.

The dominant strategy is to take try and force Merciful End. Generally you start athena dash and pray for ares and athena as soon as possible. You need Ares attack + Athena Special and then you are eligible for the duo boon. Next priority is impending doom from ares and defensive boons / the legendary from Athena. Artemis cast will enable Deadly Reversal for the ultimate high roll, but if you don't have Merciful End yet, this may not be desirable as it can reduce your ME chances.

Note that even top runners struggle to clear the game and the number of non-ME first run clears within our community can probably be counted on one hand (and ME clears on two). In other words, this run is very hard and ME is one of the few ways to really get enough damage. You can grind it out on a worse build, but it will be exponentially harder and longer.

Aside from that, just work on your sword mechanics. Watch Vorime's run for some great examples of high skill swordplay, but remember you can play safer than him as he is speedrunning. He has gone over some sword mechanic tips on a few of his streams, so definitely stop by there for advice etc.

Lastly, feel free to join our discord in the sidebar. We have a fresh file channel that is super supportive and helpful, and even if you view this more of a challenge run than a speedrun, we will root for you all the same :D