Nanopore sequencing and assembly of a human genome with ultra-long reads

By Miten Jain, Sergey Koren, Karen H. Miga, Joshua Quick, Arthur C. Rand, T. Sasani, John R. Tyson, A. Beggs, A. Dilthey, Ian T. Fiddes and 16 more. Nature Biotechnology, 2017

1,714 citations recorded by the source corpus.

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/d636ec287c6c24f56f3274ed6f3c198e7b8579e6

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Citations in the corpus

DNA sequencing at 40: past, present and future (2017, positive): “Indeed, both PacBio and nanopore sequencing have demonstrated the detection of native covalent modifications, such as methylation 64,65 .”
Sequencing DNA with nanopores: Troubles and biases (2021, background): “It offers many interesting features, including long read sequencing (the mean read length often exceeds 10 kb, and maximal read length now reaches up to 880 kb [2]), a real-time analysis and a low initial investment.”
Polypolish: Short-read polishing of long-read bacterial genome assemblies (2021, neutral): “Short reads from Illumina platforms do not suffer from the same errors in homopolymer sequences as long reads [5].”
Time- and memory-efficient genome assembly with Raven (2021, background): “Canu was not run on human datasets because of its long running time, but we found assemblies in other publications 5,23 .”
Falco: high-speed FastQC emulation for quality control of sequencing data (2019, neutral): “One dataset was down-loaded from the Whole Human Genome Sequencing Project 17 .”
Readfish enables targeted nanopore sequencing of gigabase-sized genomes (2020, methodology): “SNPs were compared with a 35x WGS NA12878 reference set recalled using the same guppy basecaller model 12 .”
Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome (2019, methodology): “PB, PacBio; ONT, Oxford Nanopore. b , Predicted contiguity of a human assembly based on ability to resolve repeats of different lengths ( x axis) and percent identities (colored lines) 21 .”
Current challenges and best-practice protocols for microbiome analysis (2019, methodology): “platform, the Oxford Nanopore MinIONTM DNA sequencer [150].”
Long-read human genome sequencing and its applications (2020, neutral): “Only 500 Mb to 2 Gb of ultra-long-read data are typically produced per flow cell with the MinION and the GridION, with a maximum throughput of 2.5 Gb (ReFS 34,35 ) .”
The complete sequence of a human genome (2021, background): “PacBio’s multi-kilobase, single-molecule reads (10) proved capable of resolving complex structural variation and gaps in GRCh38 (8, 11), while Oxford Nanopore’s >100 kbp “ultra-long” reads (12), enabled the first complete assemblies of a human centromere (ChrY) (13) and chromosome (ChrX) (14).”
Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes (2020, methodology): “Shasta was designed to be orders of magnitude faster and cheaper at assembling a human-scale genome from nanopore reads than the Canu assembler used in our earlier work 18 .”
HiCanu: accurate assembly of segmental duplications, satellites, and allelic variants from high-fidelity long reads (2020, methodology): “This is 30-fold less than recent Oxford Nanopore assemblies that required more than 100,000 CPU hours (Jain et al. 2018b; Shafin et al. 2020).”
Telomere-to-telomere assembly of a complete human X chromosome (2019, positive): “High molecular weight DNA from CHM13 cells was extracted and prepared for nanopore sequencing using a previously described ultra-long read protocol 1 .”
TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads (2020, positive): “As a comparison, the de novo assembly for 30 × coverage of long reads requires ∼ 40,000 CPU hours for ONT and ∼ 62,000 CPU hours for PacBio [34].”