Benchmarking of sequencing technologies defines optimal strategies for genetic variants detection in a human genome

Advances in sequencing technologies continue to improve the resolution and completeness with which human genetic variation can be characterized. Short-read sequencing remains widely used due to its high base accuracy, throughput, and cost efficiency; however, its limited ability to resolve repetitive and structurally complex regions has accelerated adoption of long-read sequencing platforms, including those from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We systematically compared sequencing technologies and variant calling pipelines for small variants and structural variants across diverse genomic contexts and sequencing depths. Short-read sequencing combined with DRAGEN achieved high accuracy for single-nucleotide variants (SNVs) and indels in well-mapped and moderately complex regions but showed reduced sensitivity and completeness for structural variant detection. In contrast, long-read sequencing platforms demonstrated clear advantages in detecting structural variants and resolving small variants in difficult genomic regions, although challenges remain in specific indel-prone sequence contexts. Among long-read pipelines, PacBio Revio with DeepVariant achieved the highest SNV and indel accuracy genome-wide, while ONT R10 with DeepVariant performed particularly well in clinically relevant loci. Structural variant detection was dominated by long-read optimized callers, with SVIM and Sawfish performing best for PacBio, and Sniffles2 and CuteSV2 for ONT, consistently outperforming short-read-based methods across variant classes and sizes. Coverage analyses indicated that long-read sequencing reached accuracy saturation between 20 × and 45 × , whereas short-read sequencing required more than 60 × coverage to approach maximal genome completeness. These results provide practical guidance for platform and pipeline selection. Long-read sequencing enables more comprehensive detection and resolution of structural variants and variation in complex genomic regions, while short-read sequencing remains a cost-effective and scalable solution for high-throughput genotyping and clinically focused applications.