Integrating next-generation sequencing with biochemical screening to optimize first-tier newborn screening systems.

Newborn screening (NBS) is a critical public health initiative aimed at the early detection of congenital disorders. The emergence of next-generation sequencing (NGS) holds the potential to expand the spectrum of diseases detectable at birth. To evaluate the feasibility of integrating NGS into existing biochemical screening workflows, we conducted a prospective pilot study involving 1,810 newborns. In parallel with routine biochemical screening, we employed two NGS strategies—multiplex PCR-NGS (M-NGS) and target-capture NGS (T-NGS)—to compare their performance. A total of 32 cases were confirmed via NGS or biochemical methods. Notably, NGS identified 17 cases missed by biochemical screening, including 15 cases of hereditary deafness and 2 of congenital hypothyroidism. T-NGS demonstrated high sensitivity [96.88% (31/32); 95% CI: 83.8–99.4%], specificity [96.97% (1759/1814); 95% CI: 96.0–97.7%], and a positive predictive value (PPV) of 36.05% [31/86; 95% CI: 26.6–46.7%]. In contrast, M-NGS exhibited lower sensitivity [43.75% (14/32); 95% CI: 27.2–61.5%] but higher specificity [99.33% (1772/1784); 95% CI: 98.8–99.6%] and PPV [53.85% (14/26); 95% CI: 35.5–71.2%]. T-NGS also detected a broader range of variants, with GJB2 (c.109G > A, c.235del) and DUOX2 (c.1588 A > T) being the most prevalent. Furthermore, significant differences in biochemical markers were observed between carriers and non-carriers of SLC22A5, ASS1, and MCCC1 after adjusting for birth weight and gestational age. Integrating NGS into the biochemical NBS framework significantly enhances diagnostic capacity. Our findings suggest that T-NGS is a more effective screening approach than M-NGS in this context.