What is ambient RNA and how can it affect cell-type annotation?

Why

During dissociation, fragile cells can rupture, releasing transcripts into the suspension. Those molecules are captured in droplets independent of the intact cell type. For example, a T-cell droplet may receive hemoglobin transcripts from lysed erythroid cells.

A naïve annotation algorithm could mistake this for a rare mixed-lineage population or a novel T-cell state. Diagnose contamination using empty droplets, implausible marker patterns, sample conditions and estimates of background RNA composition. Tools such as SoupX or alternative decontamination approaches may reduce it, but adjustment can also subtract real low-abundance expression when assumptions are wrong.

Document whether the raw droplet matrix was available and whether a plausible control population supports the inferred contamination profile. Compare annotations before and after correction and validate rare states with protein measurements or independent libraries. A computational correction should be treated as a sensitivity analysis, not proof that every transcript belongs to a particular cell.