Proper study design is crucial for obtaining meaningful results and reaching correct conclusions in scientific investigations. How to apply key study design principles to next generation sequencing (NGS) can be unclear and is sometimes overlooked by researchers who are new to this technology. A proper study design in NGS studies can be achieved with awareness of factors that can influence sequencing results and by taking the necessary steps to limit their biasing assessment. These steps include using simultaneously or similarly sequenced controls, randomising, determining the necessary sequencing depth, and performing a power calculation to determine the necessary sample size for testing hypotheses at significance levels adjusted for multiple comparisons. Applying study design principles to NGS studies will increase the likelihood of identifying genetic factors associated with human traits and diseases.