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.
Introduction
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.
Table 1: HG002 cell lines were sequenced with guppy v6.3.2 in SUP at 400 bps using Ligation Sequencing Kit V14, and aligned to the hg19 reference assembly. Variant calling was then performed with 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 with guppy v6.3.2 in SUP at 400 bps using Ligation Sequencing Kit V14 (Table 1). Structural variants were called and evaluated against the Genome In A Bottle consortium’s HG002 SV truthset.
Nanopore technology is highly scalable. Large plant and animal genome SV surveys can be performed to high depth of coverage on the powerful PromethION device, or SVs in smaller genomes can be thoroughly assessed on the portable Flongle and MinION, whilst the GridION offers the flexibility to scale up or down to match your experimental goals. 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 targeted sequencing. 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
Challenges in germline variant interpretation for the molecular diagnosis of disease
‘Nanopore sequencing is able to detect almost all of the variants in the cases that we sequenced’.
Germline variants in cancer susceptibility genes contribute to 10–12% of all cancers. Dr Katherine Dixon (University of British Columbia) performed nanopore sequencing of clinical research samples from individuals with a known or suspected susceptibility to hereditary cancer. The genetic architecture underlying cancer susceptibility is relatively complex but Katherine and her team found with nanopore sequencing, they were able to characterise pathogenic genetic and epigenetic variation and define haplotypes associated with founder variants, revealing unforeseen allelic heterogeneity at the loci of recurrent deletions.
In research samples from familial tumours, integrating long-read germline genome sequencing and short-read tumour DNA and RNA sequencing implicated specific cancer pathways in disease. These findings suggest the potential of long nanopore sequencing reads to provide insights into the natural history, functional consequences, and clinical significance of disease-causing genetic variation.
Sequencing workflow
How can I best call structural variation with Oxford Nanopore?
A whole human genome survey can be achieved by sequencing on 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.
Discover more about the advantages of nanopore long sequencing reads for analysing structural variation.
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Structural variation detection: In action
For high-throughput whole genome sequencing and structural variation detection across large genomes, we recommend the following:
Find out more about our lower-throughput sequencing platforms, including MinION Mk1B and Mk1C.
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