Spike-in controls

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Spike-in controls are molecules added in known amounts to a biological sample. They act as an internal reference to help quantify the molecules of interest more accurately and to compare data across samples and experiments. In sequencing, spike-ins help monitor and correct for biases that happen during sample processing, such as extraction, library preparation, and sequencing.

What spike-ins are
- They can be RNA, DNA, proteins, or metabolites. They can be synthetic or exogenous materials not originally in the sample.
- In sequencing, spike-ins often come from the genome of a different species (for example, fruit fly or plant DNA/RNA) so they can be distinguished from native molecules.
- They are added early in the workflow, during or right after lysis or extraction, and go through the same steps as the native material.

Why they’re useful
- Because their starting amounts are known, the measured amounts after processing reveal technical losses or biases.
- They help adjust the data so the results reflect true biology rather than technical variation.
- They improve data quality and consistency across samples and experiments.

How they’re used in analysis
- After sequencing, you count the spike-in reads along with the endogenous reads.
- Normalization methods use these counts to create sample-specific scaling factors.
- If a sample has fewer spike-in reads than expected, endogenous counts are scaled up to compensate; more advanced methods use regression or factor analysis across multiple spike-ins.

Common approaches
- Reference-adjusted reads per million (RRPM) and other spike-in–based scaling methods.
- Some methods compare observed spike-in counts to expected counts or use total spike-in reads per sample.

Other types and settings
- Spike-ins aren’t limited to sequencing. In proteomics and metabolomics, people use stable isotope–labeled peptides (AQUA peptides), purified proteins, endogenous or non-endogenous metabolites, and other small molecules at known amounts for quantification and normalization.
- Spike-ins can be paired with unique molecular identifiers (UMIs) to boost sensitivity and accuracy in sequencing.

In short, spike-in controls provide a reliable yardstick to detect and correct technical variation, helping to produce more accurate and comparable results.