Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

Abstract

The combination of ultra-long Oxford Nanopore (ONT) sequencing reads with long, accurate PacBio HiFi reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely-studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C" chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the ultra-long reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and has the potential to provide a single-instrument solution for the reconstruction of complete genomes.

Telomere-to-Telomere Phased Genome Assembly Using HERRO-Corrected Simplex Nanopore Reads

“…Additionally, we calculated yak 13 ǪVs for the I002C reads corrected at different input coverages, finding that ǪVs increased as coverage increased (Supplementary Table 9). In the table, we also added an evaluation of the HG002 trio Verkko assembly from Cheng et al paper 15 , where they combine the HiFi and UL dataset and the HG002 trio Verkko assembly from Koren et al 2 ., where they combine the Duplex and UL dataset. The results are comparable, showing the power of correction of nanopore reads.…”

“…They used an UL dataset, extracting only reads longer than 100kbp. For comparison on the HG002 genome when Duplex + UL reads are used, we added results evaluated from assembly from Koren et al's recent preprint 2 . All assembly evaluations were done on sca?olds whenever sca?olds were produced.…”

“…Achieving telomere-to-telomere phased genomes requires using several different technologies, primarily high-quality long reads such as HiFi reads produced by Pacific Biosciences sequencers or Duplex nanopore reads (shown in 2,3 ) produced by Oxford Nanopore Technologies (ONT), and ultra-long nanopore read. In addition, for successful phasing, either trio binning using parental reads 4 or cross-linking technologies such as Hi-C, Omni-C or Pore-C is required.…”

Benchmarking of bioinformatics tools for the hybrid de novo assembly of human whole-genome sequencing data

“…Therefore, future studies using more accurate ONT basecallers could lead to significant improvements in genome assemblies. Additionally, in recent years, ONT protocols and reagents have been enhanced, enabling the production of ultra-long reads (Jain et al 2018) and duplex reads (Koren et al 2024). These advancements are expected to improve accuracy and may impact the computational time necessities, which can make the use of error-correction or polishing steps using short-reads unnecessary (Nie et al 2024).…”

The Application of Long-Read Sequencing to Cancer

“…The latest ONT Q20+ and PacBio HiFi protocols have transformed the sequencing design by producing long reads with base accuracies exceeding 99.9% [110]. The ONT Duplex protocol, a high-accuracy technique that involves sequencing both strands of DNA, was recently used to assemble a human genome with a base accuracy greater than 99.999% (Q50) and near-perfect continuity [111]. Both TGS technologies have their advantages.…”