Unraveling the potential of short and long read sequencing for human genome profiling

Background: Short read sequencing technologies have dominated the field of human whole genome sequencing in the past years in terms of cost, throughput, and accuracy. However, thanks to recent technological evolution, long read approaches have become increasingly competitive and complementary to short reads. With the gap in the cost per genome closing slowly between both approaches, long reads might replace short read sequencing in future research and clinical applications. Still, comprehensive evaluation is necessary to conclude on the performance and general advantages of each technology. Results: In this study, we compared the latest chemistries of major suppliers of short and long read technologies: Illumina short reads, Illumina Complete Long Reads (ICLR), Pacific Biosciences HiFi reads (PacBio), and Oxford Nanopore Technologies long reads (ONT). Using the HG002 human reference sample and established bioinformatics guidelines, we assessed their variant calling performance against the latest available truth sets at different levels of coverage. For single nucleotide variant detection, all technologies were equivalent. Despite the latest improvements in chemistry, indel calling with ONT continues to lag in accuracy behind other technologies. In contrast, long reads delivered a clear advantage in structural variant detection, surpassing short reads in both accuracy and sensitivity. The hybrid ICLR approach achieved intermediate performance, narrowing the gap between short and long read sequencing. Furthermore, long reads enhanced haplotype-phasing resolution, enabling the phasing of over 80% of the genome. Conclusions: These findings highlight the specific strengths and limitations of recent sequencing technologies, aiding the decision-making in future research projects, technological platforms development, and clinical applications.