Next-generation sequencing as a second-tier diagnostic test for newborn screening.

Area of Science:

• Genetics
• Biochemistry
• Molecular Diagnostics

Background:

• Tandem mass spectrometry (MS/MS) is a long-standing method for newborn screening (NBS) of inherited metabolic diseases (IMDs).
• Traditional MS/MS screening faces limitations including false positives/negatives and variability due to biochemical fluctuations.
• Next-generation sequencing (NGS) is explored to enhance diagnostic accuracy in NBS.

Purpose of the Study:

• To evaluate NGS as a second-tier diagnostic test for IMDs in NBS.
• To assess the feasibility of using genomic DNA from dried blood spots for NGS analysis.
• To determine the diagnostic accuracy of NGS compared to Sanger sequencing.

Main Methods:

• Genomic DNA extraction from dried blood spots.
• Design of a 77-gene multigene panel for over 40 IMDs.
• Sequencing using the Ion Personal Genome Machine (PGM) platform.
• Comparative analysis of NGS and Sanger sequencing for 38 abnormal samples.

Main Results:

• Sufficient DNA quantity and quality were obtained from dried blood spots for library preparation.
• Sequencing data exhibited adequate coverage and depth for reliable analysis.
• NGS results demonstrated complete concordance with Sanger sequencing findings for all tested samples.

Conclusions:

• Genomic DNA from dried blood spots is suitable for NGS, yielding dependable results.
• NGS can serve as an effective second-tier diagnostic tool in newborn screening for IMDs.
• The Ion PGM platform facilitates molecular diagnosis of IMDs when paired with appropriate gene panels.