Herein, we designed and evaluated the clinical utility of a tNGS panel (NBS_LSDs) to simultaneously screen six genes (GBA, GAA, SMPD1, IDUA1, GLA, GALC) whose mutations are responsible for a group of six LSDs (MPSI, Pompe, Krabbe, Fabry, Gaucher, and Niemann Pick A-B diseases) that are a candidate for inclusion in NBS programs. This evidence concerns the gene GLA and mucopolysaccharidosis type 1.