Targeted sequencing with nanopore technology
Enabling the generation of any length of sequencing read — from short to ultra long — nanopore technology expands the capabilities of targeted sequencing approaches beyond the analysis of single nucleotide variants (SNVs), to include high-coverage characterisation and phasing of structural variants (SV), repetitive regions, and base modifications. Real-time data streaming provides immediate access to results and underpins Oxford Nanopore's unique on-device enrichment method of adaptive sampling.
What is targeted sequencing?
Targeted sequencing involves the enrichment of target DNA/RNA molecules or depletion of unwanted molecules. It is a valuable method of generating sufficient depth of coverage for specific regions of interest, for informative and cost-effective analysis. By dedicating more sequencing time to regions of interest, their depth of coverage can be greatly increased. This can significantly reduce the number of sequencing libraries and runs required, and reduce the data analysis burden, for a quicker and more cost-efficient workflow.
While traditional methods utilise PCR-based approaches for enrichment, amplification-free target enrichment techniques, such as adaptive sampling, combined with direct nanopore sequencing allows the identification of epigenetic modifications alongside the nucleotide sequence — further expanding the utility of targeted sequencing.
Combining innovative enrichment methods with real-time sequencing, nanopore technology provides significant advantages over conventional targeted sequencing methodologies.
Why nanopore technology for targeted sequencing?
Nanopore technology offers a variety of targeted methods including amplicon-based approaches or PCR-free approaches such as Cas9 enrichment and adaptive sampling.
Using nanopore targeted sequencing, you can:
- Cost-effectively screen large and targeted regions of interest for known and novel variants
- Characterise large genomic regions and entire genes in single reads
- Choose your enrichment strategy — PCR, adaptive sampling, or CRISPR/Cas9
- Detect base modifications using direct, amplification-free sequencing of native DNA
- Analyse data in real time for immediate access to results
- Resolve structural variants, repetitive regions, SNVs, and phasing
One platform — comprehensive analysis
Experimental approach to targeted sequencing
A wide range of library preparation kits are available to suit targeted sequencing requirements. Amplification-free kits allow direct, targeted sequencing of native DNA, eliminating the potential for PCR bias and enabling the detection of base modifications (e.g. methylation) alongside the nucleotide sequence. Amplification-based kits are also available to enable sequencing of the entire 16S gene.
| Application-free, native DNA sequencing and retained base modification | Amplification-based for low DNA amounts or quality | ||
|---|---|---|---|
| Ligation Sequencing Kit | Cas9 Sequencing Kit | 16S Barcoding Kit | |
| Preparation time | 60 min | 110 min | 10 min + PCR |
| Input requirement | 1,000 ng gDNA or 100–200 fmol amplicons | 1–10 µg gDNA | <10 ng gDNA |
| Fragmentation | Optional | Not required | Not required |
| Read length | Equal to fragment length | Equal to fragment length | Full length 16S gene (~1.5 kb) |
| Typical throughput | 3/3 | 1/3 | 2/3 |
| Multiplexing options | 24 plex, 96 plex | - | 24 plex |
| Methylation included | Yes | Yes | - |
| Overview | Optimised for output; retains base modifications; control over read length | Target specific regions of 5 – 20 kb; up to 100 target sites in a single assay | Simple and rapid; full-length 16S gene provides higher resolution identification |
| Buy now | Buy now | Buy now | |
Get started with targeted sequencing
Get best practice recommendations to optimise your end-to-end targeted sequencing workflow in our Getting started guide.
Which device for targeted sequencing?
From portable, yet powerful Flongle and MinION devices through to the flexible GridION device and high-output PromethION devices — scale your sequencing to match your specific targeted sequencing requirements.
- Theoretical maximum output. Assumes system is run for 72 hours (or 16 hours for Flongle) at 420 bases / second. Actual output varies according to library type, run conditions, etc. TMO noted might not be available for all applications or all chemistries.
MinION
The portable MinION can be run from a laptop, whilst the MinION Mk1C integrates an LCD display and onboard compute for all-in-one sequencing and analysis inside or outside the lab. These are ideal for sequencing single targets or smaller panels.
Combining up to 48 independently addressable, high-capacity flow cells with powerful, integrated compute, PromethION 48 delivers flexible, on-demand access to terabases of sequencing data.
Flexible, large-scale sequencing using up to 24 independent, high-capacity flow cells.
PromethION 2 devices maintain the flexibility associated with the PromethION 24 and PromethION 48 devices, but in a compact, accessible form factor.
A compact benchtop device offering powerful integrated compute. Run multiple projects on a single device using five independent MinION Flow Cells and sample multiplexing.
All the benefits of real-time nanopore sequencing in a pocket-sized, USB-powered device.
A powerful, portable, and affordable all-in-one sequencing and analysis device.
Adapting MinION and GridION to run our lowest cost flow cells — ideal for smaller or routine assays.
Automated sample extraction and library preparation — use predefined or custom protocols.
Analysis techniques for targeted sequencing
A range of pipelines and tools are available for the analysis of nanopore targeted sequencing data, including workflows for SV calling, 16S-based taxonomic classification, and human exome alignment. Bioinformatics pipelines are available for many applications, such as the assessment of Cas9 targeted sequencing data. Best practice analysis pipelines are available with EPI2ME.
Find out more about analysing nanopore targeted sequencing data.
Featured targeted sequencing workflow
For targeted analysis of CpG regions in the human genome using reduced-representation methylation sequencing (RRMS), we recommend the following:
Inspiration for targeted sequencing
Discover more about applying nanopore targeted sequencing to your organism and genomic variants of interest.
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