Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA samples before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA samples, followed by the bioinformatic steps required to separate sequin- and sample-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1-4 d and the bioinformatic steps (which can be performed with the provided example data files) take an additional day.