Genome assembly

High-quality genome assemblies are crucial for their use as reliable reference sequences. However, the short reads produced by legacy sequencing technologies lead to highly fragmented, incomplete assemblies. Short reads cannot span important genomic regions such as repeats and structural variants (SVs), resulting in incorrect assembly. In contrast, nanopore technology can deliver long and ultra-long sequencing reads (current record >4 Mb), that can span complex genomic regions, enabling the generation of highly contiguous reference-quality genome assemblies.

Using nanopore sequencing alone, it is now possible to generate complete telomere-to-telomere (T2T) assemblies, including highly accurate Q50 whole human genomes. Register your interest in our T2T product bundle.

  • Generate highly contiguous reference-quality genome assemblies with long and ultra-long reads
  • Resolve repeats and SVs
  • Explore epigenetic modifications and eliminate bias through direct sequencing of native DNA
  • Scale to your requirements — from small microbial genomes to large plant genomes

Generate highly contiguous genome assemblies using long nanopore reads

Large SVs, repeat sequences, and GC-rich regions are challenging to accurately characterise with short-read sequencing technology, and the resulting genome assemblies tend to be fragmented due to the lack of read overlap. Nanopore technology routinely generates sequencing reads that are tens of kilobases in length and is also capable of sequencing ultra-long libraries (i.e. read N50 of >100 kb; Figure 1). The greater overlap between ultra-long reads enables easier de novo genome assembly. The longest DNA fragment sequenced to date using nanopore technology is 4.2 Mb, which was achieved using the Ultra-Long DNA Sequencing Kit. The long-read capability of nanopore sequencing not only enables accurate delineation of complex genomic regions such as repeats and SVs, but also the sequencing of smaller microbial genomes in single reads — negating the need for assembly entirely (see poster).

Figure 1. Nanopore sequencing delivers long and ultra-long read lengths that can span complex genomic regions, enabling the generation of highly complete and contiguous genome assemblies.

Tomato genome assembly metrics

Table 1. Comparison of tomato genome (Heinz 1706) assemblies. The nanopore sequencing assembly was generated using 46x duplex and 42x ultra-long simplex data, while the public SL5.0 assembly was generated using a range of technologies, including an alternative long-read capable sequencing platform plus chromosome conformation capture. Long nanopore reads enabled the generation of a full telomere-to-telomere reference genome for this organism, with the 14 contigs comprising the 12 chromosomes, plus the chloroplast and mitochondrial genomes. The nanopore sequencing data added an additional 16.7 Mb of sequence data compared with the public reference genome, with a consensus accuracy of Q51.8 (>99.999% accurate). Data kindly provided by Alexander Wittenberg, Keygene, Netherlands. Watch the video.

Comprehensive genomic analysis, including direct detection of modified bases

A common metric for assessing genome assembly quality is contig N50 — the length at which half of the nucleotides in the assembly belong in contigs of this length or longer. The use of long nanopore reads delivers significantly higher N50 values than those provided by alternative sequencing technologies, enabling the generation of more complete and more contiguous genome assemblies (Table 1). In addition, using Pore-C, a complete, end-to-end workflow for nanopore sequencing-based chromosome conformation capture, large genome assemblies can be further scaffolded and corrected. Long nanopore reads also simplify haplotyping, enabling the resolution of compound heterozygosity and parental origin. Furthermore, nanopore sequencing does not require amplification, allowing the direct detection of base modifications (e.g. methylation) alongside the nucleotide sequence for even more comprehensive genomic analyses.

Case study

Making telomere-to-telomere genome assemblies accessible

In this presentation, Sean McKenzie discusses how the latest basecalling accuracy enhancements delivered by Oxford Nanopore makes telomere-to-telomere (T2T) genome assemblies accessible to every lab, using a single PromethION device.

By combining data derived from ultra-long nanopore sequencing reads, Pore-C chromatin conformation, and polishing using the Assembly Polishing Kit, highly complete, haplotype-resolved Q50 human genomes can be reconstructed using an automated analysis pipeline, without the need for manual curation. This is now available to order as a bundle for selected customers participating in the Early Access Programme.

Case study

Capturing global genomic diversity in the human pangenome

The Human Pangenome Reference Consortium (HPRC) aims to develop a human pangenome assembly that better represents human genomic diversity than current single reference genomes. Initial sequencing of 47 diverse human genomes using the PromethION device revealed how nanopore sequencing requires less coverage to achieve the same level of accuracy as an alternative long-read capable sequencing technology. The team further demonstrated how long nanopore reads enabled the inclusion of SVs that would previously have been missed in short-read studies.

Sequencing workflow

How do I assemble genomes using nanopore sequencing?

Oxford Nanopore provides a range of sequencing devices suitable for any sized genome assembly project, from small individual microbial genomes to high-throughput, population-scale sequencing of large genomes.

For best practice advice on genome assembly, view our getting started guides for microbial sequencing and whole-genome sequencing of large genomes. These guides provide a step-by-step overview of the entire sequencing workflow — from selecting the right nanopore sequencing device through to sample preparation, sequencing, and data analysis. Our best practice workflows for human, microbial, and metagenomic genome assembly provide structured, recommended workflows for assembling genomes using nanopore sequencing technology.

Looking to perform microbial genome assembly?

View our simple, end-to-end workflows for microbial genome assembly.

Get started

High-throughput assembly of large genomes

For high-throughput sequencing and assembly of large and complex genomes, such as those of humans, animals, and plants, we recommend the following:

Ligation Sequencing Kit

PromethION 24 or 48

Flye + Medaka


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