Common issues & fixes
| Symptom | Likely cause | Fix |
|---|---|---|
| Uneven read distribution | Inconsistent input normalization | Normalize inputs accurately first; the prep is only as even as the input. |
| Low pool yield | Over-dried beads; old ethanol | Make ethanol fresh; shorten bead dry time. |
| Adapter dimers | Index:input ratio too high | Reduce adapter/index concentration for low inputs; tighten SPRI ratio. |
| Index collisions | Reused indexes across plates | Use unique dual indexes and track index assignments. |
When not to automate this
For a handful of high-input samples, a standard library prep may be simpler. While you're optimizing a novel input type, develop manually first — automation amplifies a stable process, it won't fix an unstable one.
Verification: this protocol is simulation-verified — confirmed to run in the simulator but not yet run on hardware by RoR. Validate before using with critical samples.