Analytical validation of germline small variant detection using long-read HiFi genome sequencing.

Area of Science:

• Genomics
• Molecular Biology
• Clinical Diagnostics

Background:

• Short-read sequencing is standard for clinical genetic testing but struggles with complex genomic regions.
• Long-read sequencing technologies, particularly HiFi chemistry, offer potential for improved variant detection.
• Advances in long-read sequencing necessitate analytical validation for clinical applications.

Purpose of the Study:

• To analytically validate long-read HiFi genome sequencing for small variant detection (SNVs/indels <50 bp).
• To compare the accuracy of HiFi sequencing against short-read sequencing.
• To assess the performance of HiFi sequencing across diverse genomic regions and specimen types.

Main Methods:

• HiFi genome sequencing was performed on reference materials and clinical specimens.
• Accuracy was evaluated by comparing HiFi data to short-read data and Genome in a Bottle (GIAB) standards.
• Performance metrics included recall, precision, and F1-score across difficult and non-difficult genomic regions.

Main Results:

• HiFi sequencing achieved >99.9% recall and >99.7% precision for SNVs, and >99.8% recall and >99.1% precision for indels in high-confidence regions.
• HiFi sequencing outperformed short-read sequencing in overall SNV/indel F1-score accuracy.
• High concordance (>99.8% for SNVs, >98.6% for indels) was observed across reproducibility and specimen type assessments.

Conclusions:

• Long-read HiFi genome sequencing provides highly accurate and robust detection of small variants (SNVs/indels).
• The technology demonstrates superior performance compared to short-read sequencing, especially in challenging genomic areas.
• These findings support the clinical implementation of long-read HiFi genome sequencing for diagnostic testing.