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

By Aaron M. Wenger, P. Peluso, W. Rowell, Pi-Chuan Chang, R. Hall, G. Concepcion, Jana Ebler, Arkarachai Fungtammasan, Alexey Kolesnikov, Nathan D. Olson and 18 more. Nature Biotechnology, 2019

1,512 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/4a18c4d19783c0c4d0604731fb6fc57ec9025f03

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Citations in the corpus

NextDenovo: an efficient error correction and accurate assembly tool for noisy long reads (2024, background): “However, they generally have an average length of ~ 15 kilobases (kb) and hence are unable to span long tandem or highly homologous multi-copy repeats, which occur widely throughout large genomes, but very specifically in some regions such as centromeres [3, 6].”
Automated assembly scaffolding elevates a new tomato system for high-throughput genome editing (2021, neutral): “1% error), and can produce contiguous draft genome assemblies [11].”
Opportunities and challenges in long-read sequencing data analysis (2020, background): “With the potential for accurately assembling and re-assembling genomes [17, 29–32], methylomes [33, 34], variants [18], isoforms [35, 36], haplotypes [37–39], or species [40, 41], tools to analyse the sequencing data provided by long-read sequencing platforms are being actively developed,…”
Detection of mosaic and population-level structural variants with Sniffles2 (2024, neutral): “Sniffles2 remains open source (MIT license) and is available at https://github.com/fritzsedlazeck/Sniffles.”
MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio high fidelity reads (2023, positive): “In the emerging era of reference genomics, where chromosomally-complete assemblies are sought, PacBio HiFi reads are a key data type [7].”
Automated assembly scaffolding using RagTag elevates a new tomato system for high-throughput genome editing (2022, positive): “HiFi reads average 15 kbp in length, are highly accurate (~0.1% error), and can produce contiguous draft genome assemblies [10].”
DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing (2023, positive): “We downloaded 9 SMRT cells of HG002 CCS raw subreads in 15Kb, 20Kb, and 24 Kb insert sizes from Baid et al. 26 and Human Pangenome Reference Consortium 23 .”
Structural variant calling: the long and the short of it (2019, positive): “Nevertheless, multiple papers have re-ported a significant improvement in precision and recall for SV calling using long reads compared to short-read mapping approaches [2, 5, 19, 82, 97 – 99].”
The complete sequence of a human genome (2021, methodology): “Here, we describe the construction, validation, and initial analysis of a truly complete human reference genome and discuss its potential impact on the field.”
The design and construction of reference pangenome graphs with minigraph (2020, background): “iting factor in our understanding of genetic variations. Meanwhile, the advances in long-read sequencing technologies make it possible to assemble a human individual to a quality comparable to GRCh38 [1,5]. There are already a dozen of high-quality human assemblies available in GenBank [6]. Properly”