A comprehensive assessment of Next-Generation Sequencing variants validation using a secondary technology

Area of Science:

• Genomics
• Clinical Diagnostics
• Bioinformatics

Background:

• Next-generation sequencing (NGS) innovations enhance data accuracy.
• Variant validation is costly and time-consuming, hindering clinical applications.
• The necessity of validating all NGS variants requires comprehensive assessment.

Purpose of the Study:

• To evaluate the necessity of validating next-generation sequencing (NGS) variants.
• To determine if high-quality NGS variants require secondary confirmation.
• To inform clinical diagnostic workflows regarding NGS variant reporting.

Main Methods:

• Collected validation data for 7,601 NGS variants across 1,045 genes from 5,190 clinical samples.
• Utilized targeted capture panels and NGS chemistries for variant sequencing.
• Employed Sanger sequencing and mass spectrometry genotyping for variant validation.

Main Results:

• 6,939 high-quality NGS variants (≥35× depth, ≥35% heterozygous ratio) were 100% confirmed.
• 5,775 heterozygous, 760 homozygous, and 404 hemizygous variants were identified.
• Mass spectrometry genotyping validated 1.5% of NGS variants, indicating high concordance.

Conclusions:

• High-quality variants from well-validated NGS workflows can be reported directly.
• Eliminating secondary validation reduces cost and turnaround time in clinical diagnosis.
• This approach supports the broader clinical implementation of NGS technologies.