What's already known about this topic? A reliable result from non‐invasive prenatal testing depends on sufficient amount of fetal DNA and sequencing reads. A common fixed lower cutoff value of fetal fraction increases the incidence of test failures compared with study‐specific cutoff values. It is not straightforward to define study‐specific lower cutoff values of fetal DNA and sequencing reads. What does this study add? The study examined the relationship between Z scores and fetal fractions and sequencing reads as well as the combination of cutoff values between Z score, fetal fraction, and sequencing reads in 35 women carrying a trisomy 21 fetus in order to define the optimal study‐specific cutoff values and possibly decrease the test failure rate. This underscores the importance of evaluating the correlation between Z score and fetal fraction and optimal combination of cutoff values when assessing non‐invasive prenatal testing results, suggesting that each laboratory should define its own correlation and optimal combination of cutoff values, because those are study specific. © 2017 John Wiley & Sons, Ltd.