Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening

Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing–based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.