Duplex sequencing

Duplex sequencing is recommended for resolving the most challenging genomic regions, enabling construction of the most complete genome assemblies possible.

What is duplex sequencing?

Duplex sequencing reads the second DNA strand, in addition to the first strand, to further increase single molecule accuracy — with the two orthogonal signals providing complementary information. By combining data from both strands into one basecall, duplex sequencing generates single molecule accuracy of ~Q30.

Duplex sequencing has already demonstrated perfect reads of up to 72 kb in length and Q30+ reads at 260 kb.

Currently, High Duplex Flow Cells are in the closed early access phase. To participate in the early access programme, register below.

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PromethION High Duplex Flow Cell.

Optimising duplex sequencing

Duplex sequencing can be maximised by using the High Duplex Flow Cells in combination with an optimised protocol and EPI2ME analysis tools.

The solution is suitable for resolving the most challenging genomic regions, enabling construction of the most complete genome assemblies possible.

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Enhanced de novo assembly through consistent long and ultra-long sequencing reads

Achieve greater de novo assembly accuracy by resolving even the most complex regions of the genome with duplex sequencing, including telomeres and centromeres, while also analysing large structural variations, complex rearrangements, and repeat expansions with confidence.

The High Duplex PromethION Flow Cells consistently deliver a substantial proportion of duplex reads essential for your specific application.

More on accuracy

Comparison of read length between the output of a single standard flow cell and three High Duplex Flow Cells for two libraries of 25 kb and 50 kb. Run using Kit 14 chemistry, 400 bps at 5 kHz.

Comparison of standard PromethION Flow Cell vs. High Duplex Flow Cell output. Human genome sample (HG002) with ~30 kb read N50. Run using Kit 14 chemistry, 400 bps at 5 kHz.

PromethION High Duplex Flow Cell output

From the total output of a PromethION High Duplex Flow Cell, around 60% of the first and second strand simplex data will be compiled to generate high-quality duplex sequencing data. The remaining 40% represents unpaired simplex data.

For a typical PromethION High Duplex Flow Cell run (native human DNA) with a total output of 120 Gb, around 35 Gb will be duplex sequencing data and 50 Gb will be simplex sequencing data (see figure).

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Comparison of PromethION Flow Cell (standard) vs. High Duplex Flow Cell output.

The high accuracy and low cost of simplex sequencing [link] makes it suitable for the vast majority of applications; however, for extremely challenging genomic regions and applications, such as sequencing telomeric or centromeric repeats for telomere-to-telomere (T2T) assemblies, duplex sequencing, which offers >99.9% single molecule accuracy, may provide additional benefits.

Run condition Simplex sequencing Duplex sequencing
Basecalling mode Simplex with high Accuracy (HAC) Duplex with super Accuracy (SUP)
Recommended for High-quality analysis: genomics variants (SVs, SNVs, etc.), phasing, methylation detection, de novo assembly, etc. Highest genome completeness and high-quality analysis: large de novo assemblies,  telomere-to-telomere
Full coverage of the human reference genome Yes Yes
Telomere to telomere coverage No Yes
Recommended human genome coverage/flow cells required 30x from one PromethION Flow Cell 30x from three PromethION High Duplex Flow Cells
Typical output (human genome) (Giga bases) 90-120Gb 35Gb high duplex (first and second strand combined) + 50Gb simplex
Computation requirements ●●○○ ●●●●
Single nucleotide variant calling accuray Similar
Structural variant calling accuracy Similar
Insertions/deletions calling accuracy Similar

See how duplex is being used by the Nanopore Community:

Redefining telomere-to-telomere genome assembly strategy using the Oxford Nanopore platform

Jianjun Liu explored the use of duplex reads plus ultra-long nanopore sequencing reads to generate telomere-to-telomere data. Jianjun revealed how constructing haplotype-resolved, high-quality telomere-to-telomere genomes is achievable using nanopore sequencing alone.

Expanding studies of global genomic diversity with complete, telomere-to-telomere assembly of diploid genomes

Karen Miga and team demonstrated the use of a new innovative assembly method, Verkko, to phase and reconstruct complete human chromosomes using a combination of duplex reads and ultra-long nanopore datasets. In parallel, Karen also discussed how new automated workflows were used to assess base-level accuracy and correct structural reconstruction of repetitive regions.