Structural variation

Structural variants (SVs) are of high importance in both normal and aberrant phenotypes; however, their detection using traditional technologies is limited by their size, complexity, and position in the genome. Long PCR-free nanopore reads can span SVs end-to-end enabling unprecedented resolution of even highly complex variants — in any genomic context.

  • Structural variants (SVs) are of high importance in both normal and aberrant phenotypes
  • Amplification-free whole genome or targeted sequencing approaches — detect base modifications as standard
  • Scalable to any project size, including large population-scale studies

Sequencing structural variants with short and long reads

Structural variants (SVs) are of high significance across a broad range of fields, from clinical research into their roles in diseases such as cancer, through to identifying SVs encoding desirable crop traits in agricultural science. However, as SVs reach up to the megabase scale, many cannot be spanned by short reads; instead, they must be sequenced in short sections and reassembled. This can result in incomplete or incorrect assemblies, whilst the requirement of PCR, an intrinsic part of the short-read workflow, means that SVs in regions which cannot be amplified may not be represented at all.

With Oxford Nanopore, there is no limit to read length: single reads frequently reach hundreds of kilobases in length, with a current record of over 4 Mb. This means that even large SVs can often be sequenced end-to-end in single reads, making for simple, accurate characterisation (see below) and often removing any need for assembly. Amplification is not required, avoiding PCR bias and allowing SVs to be identified across the genome, including in repetitive or GC-rich regions, such as repeat expansions, which are inaccessible to other methods. This also enables the sequencing of intact modified bases, so that SVs and their epigenetic effects can be revealed in a single experiment.

Investigating repeat expansions
SV detection using nanopore sequencing

Table 1. Libraries from the HG002 cell line were prepared using the Ligation Sequencing Kit, and sequenced using 5 kHz MinKNOW scripts, basecalled with Dorado v0.2.5 in HAC at 400 bps, and aligned to the hg19 reference assembly. Variant calling was then performed using the latest version of Sniffles, and variants were compared against the Genome In A Bottle consortium’s HG002 SV truthset; view the Structural variation workflow for full sample-to-answer guidance.

Long nanopore reads enable calling of SVs across the genome with high precision and recall

To assess the performance of SV calling with nanopore sequencing, human (HG002) cell lines were sequenced using the Ligation Sequencing Kit (Table 1). Structural variants were then called and evaluated against the Genome in a Bottle consortium's HG002 SV truth set. Nanopore technology is highly scalable: large plant and animal genome SV surveys can be performed to high depth of coverage on powerful PromethION Flow cells, or SVs in smaller genomes can be thoroughly assessed on Flongle and MinION flow cells. Compatible devices for these flow cells include the portable MinION Mk1B, through to the powerful production capabilities of the PromethION 48 which can run up to 4,992 30x human genomes per year. Targeted sequencing can also be used to enrich for SVs of interest, including PCR-free targeting of large SVs in any region of the genome using adaptive sampling — a unique on-device enrichment methodology, or Cas9-based enrichment. With simple library preparation, in as little as ten minutes, and real-time sequencing and analysis, including a dedicated SV analysis pipeline, nanopore sequencing is a powerful tool for the study of SVs.

Case study

The detection of large SVs using nanopore sequencing provides greater insight into the genetic drivers of disease

LRS [Long read sequencing] greatly enhances SV discovery over short-read NGS

Chemparathy et al. medRxiv (2023)

Using long nanopore sequencing reads, Chemparathy et al. ‘directly sequenced and properly aligned’ large SVs to the human genome, enabling them to identify a novel SV in linkage disequilibrium with single nucleotide variants associated with the risk of frontotemporal lobar dementia and Alzheimer’s disease. The SV had been missed in previous studies yet could mediate disease pathogenesis and may provide insight into the genetic drivers of such diseases.

Nanopore sequencing of large SVs
Case study

Nanopore sequencing can help better understand the genetics of platelet disorders


Nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization

Zamora-Cánovas et al. J. Thromb. Haemost. In press (2023)

To understand the genetics of inherited platelet disorders, Zamora-Cánovas et al. used the MinION to target regions of interest in the human genome using adaptive sampling — an on-device enrichment method unique to Oxford Nanopore. According to the researchers, long nanopore sequencing reads enabled the successful characterisation of novel SVs ‘using a low-cost instrument in a rapid and cost-effective manner’.

Read the publication
Sequencing workflow

How can I best call structural variation with nanopore sequencing?

Whole human genome sequencing, enabling comprehensive genome-wide SV characterisation, can be achieved using a single PromethION Flow Cell. We recommend preparing your library using the Ligation Sequencing Kit — to generate high outputs of long reads — and sequencing to 30x depth of coverage. For analysis, our SV pipeline will take you from raw data to SV calling and visualisation; view our step-by-step tutorial for instructions, or use our fully-automated EPI2ME SV calling workflow which does not require use of the command line.

Download the structural variation white paper

Discover more about the advantages of nanopore long sequencing reads for analysing structural variation.

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For high-throughput whole genome sequencing and structural variation detection across large genomes, we recommend the following:


Ligation Sequencing Kit

Analysis: wf-human-variation


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