Nathan Hammond1,3, Linda Liao1, Pun Wai Tong1, Zena Ng1, Thuy-Mi P. Nguyen2, Chandler Ho1, Yao Yang1,2 and Stuart A. Scott1,2 1Clinical Genomics Laboratory, Stanford Medicine, Palo Alto, California 94304, USA; 2Department of Pathology, Stanford University, Stanford, California 94305, USA

↵3 Present address: Influx Bio, San Francisco, CA 94124, USA

Corresponding author: sascottstanford.edu Abstract

Long-read sequencing has the capacity to interrogate difficult genomic regions and phase variants; however, short-read sequencing is more commonly implemented for clinical testing. Given the advances in long-read HiFi sequencing chemistry and variant calling, we analytically validated this technology for small variant detection (single nucleotide variants, insertions/deletions; SNVs/indels; <50 bp). HiFi genome sequencing was performed on DNA from reference materials and clinical specimen types, and accuracy results were compared to short-read genome sequencing data. HiFi genome sequencing recall and precision across Genome in a Bottle (GIAB)-defined non-difficult and difficult genomic regions (high confidence) for SNVs are >99.9% and >99.7%, respectively, and for indels are >99.8% and >99.1%, respectively. Moreover, HiFi genome sequencing outperforms short-read genome sequencing on overall SNV/indel F1-score accuracy at all paired sequencing depths, which are further stratified across 100 total GIAB-defined genomic regions for a comprehensive evaluation of performance. Of note, HiFi genome sequencing F1-scores for SNVs and indels surpass 99% at ∼15× and ∼25×, respectively. In addition, high confidence small variant concordance across all HiFi genome sequencing reproducibility assessments (two specimens, three independent sequencing data sets) are >99.8% for SNVs and >98.6% for indels, and average high confidence small variant concordance between paired blood, saliva, and swab specimens are all >99.8%. Taken together, these data underscore that long-read HiFi genome sequencing detection of SNVs and indels is very accurate and robust, which supports the implementation of this technology for clinical diagnostic testing.

Received December 9, 2023. Accepted April 11, 2025.