Nanopore Target Seq FAQs
Q1: What is nanopore targeted sequencing and how does it differ from whole-genome sequencing?
Nanopore targeted sequencing focuses on specific genomic regions of interest—such as genes, hotspots, or structural variant breakpoints—rather than sequencing the entire genome. This approach enables higher coverage of those targets, less data waste, and retention of long reads and epigenetic modifications. Whole-genome sequencing provides comprehensive coverage but is more costly and generates more data than needed for focused studies.
Q2: What are the different methods or "routes" to do targeted sequencing with nanopore technology?
There are several approaches: (1) PCR-based amplicon sequencing for well-defined small targets; (2) probe or hybrid capture panels (less common in nanopore but available via custom or third-party designs); (3) CRISPR-Cas9 enrichment, which is amplification-free and suited for structural variants and difficult genomic regions; and (4) adaptive sampling, which enriches or depletes reads in real time using software without additional wet-lab steps.
Q3: What is the difference between nanopore amplicon sequencing and other targeted sequencing methods?
Amplicon sequencing is PCR-based, cost-effective, and rapid, but it may introduce amplification bias, miss GC-rich or repetitive regions, and remove methylation information. Cas9 enrichment or adaptive sampling avoids PCR, preserves methylation, and captures structural complexity across long genomic regions.
Q4: How does nanopore Cas9 targeted sequencing compare with hybrid capture?
Cas9 enrichment is faster, simpler, and probe-free, producing long reads that resolve repeats and structural variants better. Hybrid capture is useful for broad panels, but requires probe design, more steps, and may not capture complex structural contexts as effectively.
Q5: Can nanopore targeted sequencing detect epigenetic modifications such as DNA methylation?
Yes. When using amplification-free methods like Cas9 enrichment or adaptive sampling, nanopore sequencing directly detects base modifications, including methylation, without additional chemical treatments or library preparation.
Q6: Can adaptive sampling be applied to low-input or degraded DNA?
Yes, adaptive sampling works with standard nanopore libraries. For highly fragmented or low-input DNA, such as FFPE or ancient samples, modified protocols can help improve performance, although enrichment efficiency depends on fragment size and quality.
Q7: What are the limitations or trade-offs of nanopore targeted sequencing compared to short-read or PCR-based methods?
Nanopore sequencing may have lower single-read accuracy than short-read platforms, though coverage and consensus improve results. Amplicon methods can suffer from PCR bias, while adaptive sampling efficiency depends on target size and DNA length. In contrast, nanopore sequencing delivers long reads, preserves methylation, and resolves structural variants and repeats that short-read or PCR-only approaches cannot.
Q8: What sample quality and input are required for reliable targeted sequencing results?
High molecular weight DNA is preferred, with minimal degradation, proper purity ratios (OD 260/280 ~1.8; OD 260/230 ~2.0–2.2), and sufficient concentration. Amplicon sequencing tolerates smaller or more fragmented inputs, while Cas9 enrichment and adaptive sampling perform best with longer DNA fragments and high-quality preparations.
Q9: Which nanopore device should I choose for targeted sequencing projects?
Device selection depends on scale and complexity. MinION is suitable for small panels or individual targets, while GridION or PromethION are recommended for larger panels, multiple samples, or projects requiring higher throughput. Multiplexing options, flow cell type, and read length goals also influence device choice.
Q10: How is adaptive sampling set up and what does it do for targeted sequencing?
Adaptive sampling is configured in MinKNOW by providing reference files and optional BED coordinates for target regions. During sequencing, software evaluates read starts in real time, allowing on-target reads to continue while rejecting off-target reads to free pores. This provides enrichment or depletion without extra wet-lab enrichment, with effectiveness depending on target size and input DNA quality.
Q11: How many variants can be detected reliably with nanopore targeted sequencing?
Nanopore targeted sequencing can detect SNVs, small indels, structural variants such as large deletions or inversions, repeat expansions if reads span them, and haplotypes when coverage and read length allow. Sensitivity and specificity depend on target design, sequencing depth, and DNA quality.
Q12: What bioinformatics deliverables are included in a standard project?
Typical deliverables include raw reads (FASTQ), alignments (BAM), variant calls (VCF for SNVs, indels, SVs), coverage and enrichment statistics, optional methylation or base modification files, and graphical summaries such as variant maps or structural variant plots. Annotation reports linking results to genes, known variants, or pathways are also provided.
