Utility of long-read sequencing for All of Us.

The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compare the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis reveals substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also consider the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produce the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results lead to widespread improvements across AoU.

Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk.

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase ~50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

Potential applications of nanopore sequencing for forensic analysis.

Advancements in DNA sequencing technologies are occurring at a rapid rate. Various platforms have proven useful in all aspects of health and science research, from molecular diagnostics in cancer research to spore identification in bioterrorism. In the field of forensics, one particular single-molecule sequencing platform shows promise for becoming a viable solution for small to midsize forensic laboratories. Oxford Nanopore Technologies (ONT) has developed a portable, nanopore-based sequencing instrument that has already been utilized for on-site identification of Zika and Ebola viruses, full genome sequencing, evaluation of DNA and RNA base modifications, and enrichment-free mitochondrial DNA analysis. The rapid development of this technology creates possibilities relevant to standard DNA sequencing, direct analysis of forensic samples, including blood, semen, and buccal swabs, mitochondrial DNA analysis, SNP and STR analysis, familial identification, and microbial identification for bioterrorism and geolocation. The small size of the platform, its low cost, and its requirement of only basic laboratory equipment makes this platform well suited for small laboratories wishing to begin developing expertise in sequence-based forensic analyses. Herein, we outline recent developments and applications of nanopore sequencing technologies and their potential application in forensic analysis. We address current and potential techniques in mitochondrial DNA analysis, SNP and STR typing, and microbial identification. Additionally, we discuss recent developments in library preparation and data analysis tool further streamlining the sequencing process that integrate workflows in laboratories or in remote field scenarios.