Abstract Molecule counting is central to single-cell sequencing, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes , novel RNA spike-ins containing inbuilt unique molecular identifiers that we use to identify critical experimental and computational conditions for accurate RNA counting across single-cell RNA-sequencing methods. The molecular spikes are a new gold standard that can be widely used to validate RNA counting in single cells.

Molecular spikes: a gold standard for single-cell RNA counting