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

Area of Science:

• Genomics
• Bioinformatics
• Medical Diagnostics

Background:

• Newborn screening (NBS) increasingly relies on genetic variant analysis for confirmatory testing.
• Current methods like Sanger sequencing and gene-specific NGS are expensive and not easily scalable for new NBS disorders.

Purpose of the Study:

• To develop and validate a scalable, exome sequencing-based NGS pipeline for universal application to any NBS disorder.
• To enable efficient genetic variant analysis for second-tier NBS testing.

Main Methods:

• Utilized de-identified newborn screening specimens for conditions including SCID, CF, VLCAD deficiency, and MLD.
• Employed an exome sequencing NGS pipeline with a priori analysis restriction.
• Curated and validated variants from multiple databases to support bioinformatics interpretation.

Main Results:

• The pipeline achieved 100% detection rates with in silico data sets.
• CFTR variant panel analysis correctly identified all variants, with concordance to diagnostic testing ranging from 78.6% to 100%.
• Database analysis revealed significant variability in variant overlap (3-65%) and a need for manual curation in 11% of variants.

Conclusions:

• The developed NGS pipeline is scalable and can be restricted to specific genes or expanded to whole exome analysis.
• This approach offers a flexible and universally applicable solution for genetic variant analysis in NBS.
• The pipeline supports clinical decision-making through curated variant data.