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New method improves next-generation sequencing accuracy 1000-fold

A new method has been published in Nature Genetics that offers a 1000-fold increase in accuracy compared to next-generation sequencing (NGS). Concatenating Original Duplex for Error Correction (CODEC) uses single duplex DNA sequencing to enable more precise genetic testing and reveal mutations that would be obscured by NGS errors.

Identifying single duplex mutations

Detecting low abundance mutations from single double-stranded DNA molecules (“single duplex”) is crucial in many fields including biomarker discovery, cancer evolution, somatic mosaicism, infectious diseases and aging. Single molecular sequencing technologies (PacBio and Oxford Nanopore Technologies) can keep single duplexes intact and sequence them in whole. In practice however, they lack the required accuracy and throughput. NGS can offer superior read accuracy and throughput, but it is not configured to sequence single duplexes without compromising throughput or utility.

Several methods have been developed aimed at addressing these issues, but these have been limited to either targeted panels or shallow sequencing. In contrast, CODEC adopts an alternative, hybrid approach that combines the massively parallel nature of NGS with the resolution of single-molecule sequencing (see Figure 1).

Figure 1: Overview of CODEC. a. CODEC physically links strands of each original duplex and each cluster reads an NGS library molecule with sequences of both strands. b. CODEC uses the adapter quadruplex followed by strand displacing extension. c. CODEC is compatible with both targeted sequencing and WGS. Taken from Single duplex DNA sequencing with CODEC detects mutations with high sensitivity (Bae et al., 2023).

Demonstrating the CODEC workflow

A proof of concept for the CODEC workflow was first performed to confirm that it could create the intended NGS library structure. The CODEC workflow was then applied to various types of biological analyses. These included the detection of germline and somatic mutations, tumour mutation profiling and the detection of microsatellite instability.

Mutation frequencies of 2.72×10-8 were detected in the sperm of a 39-year-old individual, as well as somatic mutations acquired with age in blood cells. CODEC also detected genome-wide clonal haematopoiesis mutations, single mutated duplexes from tumour genomes and liquid biopsies and microsatellite instability with 10-fold greater sensitivity.

Enabling precise genetic analysis

CODEC unlocks more possibilities for NGS testing by enabling more precise genetic analysis at a lower cost. Combined with the deeper biological insights gained at a single DNA molecule resolution, CODEC has the potential to deliver DNA sequencing that is also accurate, with great value for biomedical research and clinical diagnostics. Viktor Adalsteinsson, senior author of the paper, stated: “The beauty of this approach is that it’s not an overhaul of how sequencing is done. It’s not something that requires new instrumentation or capital investment — it’s a simple set of steps added into existing sample preparation workflows to improve the accuracy of DNA sequencing. This technology is enabling us to see things that we could never see before with DNA sequencing, and that’s tremendously exciting.”

More on these topics

DNA / Genetic Variants / NGS / Sequencing