Spike-in controls

Spike-in controls or spike-ins are known quantities of molecules—such as oligonucleotide sequences (RNA, DNA), proteins, or metabolites—added to a biological sample for more accurate quantitative estimation of the molecule of interest across samples and batches.^[1] Spike-ins are particularly used in high-throughput sequencing assays,^[2] where they act as an internal reference to monitor and normalize technical and biological biases introduced during sample processing such as library preparation, handling, and measurement.^[3][4][5]

Spike-ins can adjust for specific technical biases and enable accurate estimation of the endogenous molecules of interest, resulting in improved data quality and standardization across different samples or experiments. Spike-ins can be synthetic or exogenous material (not originally part of the sample). In sequencing-based assays, exogenous material is typically derived from the genome of a different species such as Drosophila melanogaster or Arabidopsis thaliana.^[6]