Common issues & fixes
| Symptom | Likely cause | Fix |
|---|---|---|
| Low library yield | Old/evaporated 80% ethanol; beads over-dried; under-amplification | Make ethanol fresh that day; shorten bead dry time; add 1–2 indexing PCR cycles if complexity allows. |
| Adapter dimers (~120 bp) | Adapter:input ratio too high; size selection ratio off | Reduce adapter concentration for low inputs; tighten the right-side SPRI ratio. |
| Broad / shifted insert size | Fragmentation time/temp off; degraded input | Recalibrate fragmentation to your kit; re-QC input integrity before the run. |
| Index hopping / cross-talk | Single-index reuse; carryover | Use unique dual indexes; fresh tips per transfer (default in this protocol). |
| Run paused mid-protocol | Vision flagged a bead/plate exception | Re-seat the flagged item as prompted; the robot resumes from that step. |
When not to automate this
If you run library prep only occasionally — a few samples a month — manual prep may still be more economical than the reagent dead-volume and setup overhead of automation. If you're still optimizing a novel chemistry where conditions change every run, develop it manually first; automation amplifies a stable process, it won't stabilize an unstable one. And for very low-input or single-cell applications, validate carefully — small volumes magnify any handling error.
Verification: this protocol is simulation-verified for a generic library-prep workflow — it is not tied to a specific kit and has not been vision-verified on hardware by RoR. Run it end-to-end with your kit and controls, and confirm QC on the first batch, before production use.