Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome

Abstract

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the ‘genome in a bottle’ (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads. High-fidelity reads improve variant detection and genome assembly on the PacBio platform.

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Affiliated Institutions

• National Institute of Standards and Technology US
• Max Planck Institute of Molecular Cell Biology and Genetics DE
• Agricultural Genomics Institute at Shenzhen CN
• Max Planck Institute for Informatics DE
• Saarland University DE
• Dana-Farber Cancer Institute US
• DNAnexus (United States) US
• Johns Hopkins University US
• Chinese Academy of Agricultural Sciences CN
• Pacific Biosciences (United States) US
• Baylor College of Medicine US
• National Human Genome Research Institute US
• Google (United States) US

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