Sample Submission GuidelinesOxford Nanopore Sequencing Services — Ultra-Long Reads, Real-Time Decisions, Direct RNA
The only sequencing technology that delivers Mb-class ultra-long reads, streams data in real time, and sequences native RNA directly — without reverse transcription or amplification. CD Genomics' Oxford Nanopore platform gives you the longest reads in genomics, the ability to stop a run when you have enough data, and direct access to RNA modifications that other platforms miss.
What we provide:
- Ultra-long reads (Mb-class) for gap-free genome assembly and complex repeat resolution
- Direct RNA sequencing — native RNA, no reverse transcription, no amplification bias
- Real-time sequencing control — monitor yield live, stop when targets are met
- End-to-end project support: library prep → sequencing → bioinformatics → publication-ready reports
Problems we solve:
- Resolve telomeres, centromeres, and segmental duplications — regions where short reads give up
- Sequence full-length isoforms and detect RNA modifications directly from native RNA molecules
- Monitor sequencing progress live and terminate runs the moment you have sufficient data
Trust: SOP-driven QC · FASTQ plus optional FAST5/POD5 · consultative study design
When Ultra-Long Reads and Real-Time Matter
If your project requires reads longer than 20 kb — to span large structural variants, close genome gaps, or resolve full-length transcript isoforms — Oxford Nanopore is your best option. Nanopore holds the record for the longest sequencing reads ever produced (exceeding 2 Mb). It is also the only platform that streams data in real time — allowing you to stop a run as soon as you have enough coverage — and the only platform that sequences native RNA molecules directly, preserving modification information that cDNA-based methods lose.
Of course, raw nanopore reads trade some per-read accuracy for these capabilities (typically Q10–20 raw, improving with latest chemistry and Dorado basecallers). For projects requiring the highest per-read accuracy, consider PacBio HiFi; for the longest read spans, real-time monitoring, and direct RNA analysis, Nanopore has no equal.
How Nanopore Sequencing Works
Nanopore sequencing passes a single DNA or RNA molecule through a protein nanopore embedded in an electrically resistant membrane. As each nucleotide transits the pore, it disrupts the ionic current in a sequence-specific manner. These current changes are recorded in real time and decoded into nucleotide sequences (A, T, C, G — or RNA bases) by a neural-network basecaller such as Dorado.
Unlike Illumina (sequencing-by-synthesis with cluster amplification) or PacBio (optical detection of fluorescently labeled nucleotides in zero-mode waveguides with circular consensus), Nanopore reads the native molecule directly. No amplification, no synthesis, and no optical measurement are involved. The raw signal carries not only base identity but also modification information (5mC, 6mA, and RNA modifications), which can be extracted bioinformatically without additional sample preparation.
Why it matters for your research:
- Ultra-long reads (exceeding 1 Mb routinely, with record reads surpassing 2 Mb). Span telomeres, centromeres, segmental duplications, and large structural variants that cannot be resolved with short or mid-length read platforms.
- Real-time data streaming. Sequencing results are generated as the molecule passes through the pore, enabling live yield monitoring, adaptive sampling (enrich or deplete target regions on-the-fly), and immediate run termination when sufficient data is collected.
- Direct RNA and native modification detection. Sequence RNA molecules without reverse transcription or amplification bias. Detect DNA methylation (5mC, 6mA) from genomic reads and RNA modifications (m6A, pseudouridine, inosine) from direct RNA reads — all from the raw signal, with no bisulfite conversion or enrichment steps required.
Our Nanopore Sequencing Services
Nanopore Full-Length Transcript Sequencing
One-line: Isoform-level transcriptomics with long reads for accurate splicing, TSS/TES, and fusion detection.
Best for: Full-length isoform discovery/quantification in coding genes; fusion calling.
Nanopore Direct RNA Sequencing
One-line: Sequence native RNA directly—retain modification signals without reverse transcription.
Best for: RNA modification research and transcriptome profiling with minimal bias.
Nanopore Amplicon sequencing
One-line: Rapid, targeted variant detection across defined loci with long amplicons.
Best for: Panel validation, hotspot screening, clone checks, small-cohort studies.
Nanopore Full Length LncRNA Sequencing
One-line: Resolve long non-coding RNA isoforms that short-read methods miss.
Best for: lncRNA structure, isoform usage, novel transcript discovery.
Nanopore Targeted Sequencing (Cas9 or Adaptive Sampling)
One-line: Focus coverage where it matters—Cas9 capture or software-driven adaptive sampling.
Best for: Locus-specific variant/methylation analysis without whole-genome cost.
Nanopore Ultra-Long Sequencing
One-line: Maximize read length (hundreds of kb to Mb-class) for assemblies and complex repeats.
Best for: De novo assemblies, large SVs, telomeres/centromeres, repeat expansions.
Pore-C Service
One-line: Long-range chromatin contacts and scaffolding using nanopore reads.
Best for: 3D genome organization, scaffolding support for assemblies.
TAIL Iso-Seq Service
One-line: Single-molecule TSS/TES mapping and poly(A) tail-length profiling.
Best for: Transcript-end biology, isoform completeness, post-transcriptional regulation studies.
Nano tRNA Sequencing Service
One-line: Full-length tRNA sequencing with direct RNA modification detection on the Nanopore platform. Complete tRNA isoacceptor and isoform profiling with capture of native modifications including m1A, m3C, m7G, pseudouridine, and other epitranscriptomic marks.
Best for: tRNA biology, modification landscape studies, tsRNA characterization, epitranscriptomic profiling.
Long-Read Metagenomic Sequencing
One-line: Resolve complex microbial communities with long nanopore reads for species-level classification, metagenome-assembled genomes (MAGs), and resistance gene profiling. Available on both PacBio HiFi and Oxford Nanopore platforms.
Best for: Complex metagenomic communities, field-deployable metagenomics, real-time pathogen surveillance.
Nanopore Full-Length cDNA Sequencing
One-line: Sequence full-length cDNA molecules for isoform-level transcript discovery. Reverse-transcribe RNA into cDNA, then sequence directly — capturing complete transcript structures from 5' end to 3' end without assembly.
Best for: Full-length transcript isoform discovery, novel transcript identification, quantification of isoform expression.
Nanopore Full-Length 16S/18S/ITS Amplicon Sequencing
One-line: Amplify and sequence full-length rRNA gene regions (16S, 18S, ITS) with Nanopore long reads for species-level and strain-level taxonomic resolution that short-read amplicons cannot achieve.
Best for: Microbial community profiling, environmental microbiome analysis, strain-level taxonomic classification.
What You'll Receive
Each Oxford Nanopore project is delivered with transparent data, detailed documentation, and reproducible QC metrics—ensuring publication-ready confidence.
Core data files (for every project)
- FASTQ (.fastq.gz) — basecalled reads (Dorado).
- Optional raw signal — FAST5/POD5 per request for modification-aware reanalysis.
- Project memo — the nanopore sequencing protocol used (library kit, flow cell/chemistry, run settings), plus software versions and parameters.
Run & QC report (per sample and per barcode)
- Yield & throughput: total reads/bases; pass/fail counts.
- Read-length metrics: N50/N90, length histograms.
- Quality: Q-score distribution, read accuracy summaries.
- Barcoding performance: assignment rates, balance across samples.
- If aligned: mapping rate, coverage uniformity/dups, on-/off-target summaries (for targeted/amplicon).
- If applicable: pore utilization over time and run notes to support study records.
Optional Analysis Outputs (Choose by Service)
| Application | Deliverables |
|---|---|
| Genomes (Standard / Ultra-Long WGS) |
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| Epigenetics (DNA Methylation) |
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| Transcript (Full-Length Transcript / lncRNA / Direct RNA / TAIL Iso-Seq) |
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| Amplicon / Targeted (Cas9 or Adaptive Sampling) |
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| 3D Genome (Pore-C) |
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Nanopore Sequencing Protocol
- Intake & QC: Qubit quant; A260/280 ~1.8–2.0, A260/230 ≥2.0. HMW gDNA for Ultra-Long; RNA RIN ≥8.
- Library prep (by application): Standard WGS; Ultra-Long (gentle HMW handling); Amplicon; Targeted (Cas9 or adaptive sampling); Full-length cDNA/lncRNA; Direct RNA; Pore-C; TAIL Iso-Seq.
- Flow cell & barcodes: Sized to yield/targets; balance barcoded inputs.
- Sequencing & control: 1–72 h typical; live monitoring; stop/extend or reload as needed; enable adaptive sampling where supported.
- Basecalling & demux: MinKNOW + Dorado → FASTQ; optional FAST5/POD5 for modification-aware reanalysis.
- Post-run QC: yield/pass-fail; read-length N50/N90; Q-score distribution; mapping/coverage & on/off-target (if aligned).
- Documentation & hand-off: protocol memo (kits/chemistry/software), QC report, scoped analysis outputs; optional review call.
Bioinformatics and Data Analysis
Our service provides comprehensive nanopore sequencing technology, bioinformatics, and applications support for Oxford Nanopore long-read data.
Standard Bioinformatics
- Basecalling & demultiplexing: Dorado/MinKNOW to produce per-sample FASTQ.
- Run QC dashboards: yield, read-length N50, Q-score distribution, barcode balance.
- Read prep: adapter/primer trimming, filtering.
- Reference alignment (optional): long-read aware mapping with coverage summaries; BAM/CRAM if requested.
- Signal retention (optional): POD5/FAST5 preserved for modification-aware reanalysis.
Advanced Analysis
- De Novo Genome Assembly — long-read first or hybrid; polished assemblies with contiguity statistics.
- Structural Variation (SV) and CNV Calling — large indels, inversions, translocations; copy-number summaries.
- Full-Length Transcript (Isoform) Analysis and Quantification — isoform discovery, expression tables, fusion detection.
- DNA/RNA Modification (Epigenetics) Analysis — research-grade 5mC/6mA; RNA-mod signal summaries for Direct RNA.
- Metagenomic Classification — taxonomic profiling; assembly support where applicable.
- (Add-ons) Targeted/Amplicon Consensus & Variant Calling, Pore-C 3D Genome Contact Maps.
Nanopore Sequencing Applications
Where Oxford Nanopore sequencing adds the most value in research :
De novo genome assembly & finishing
Span repeats and complex regions; resolve telomeres/centromeres.
Recommended services: Ultra-Long Sequencing, Standard Long-Read WGS.
Structural variation (SV) & complex rearrangements
Detect large insertions/deletions, inversions, translocations, repeat expansions.
Recommended services: Ultra-Long/Standard WGS, Targeted (Cas9/Adaptive).
Haplotype phasing & allele-specific analysis
Long molecules preserve linkage across distant variants.
Recommended services: Standard/Ultra-Long WGS.
Full-length transcriptomics (isoforms & fusions)
Identify novel isoforms, quantify usage, confirm fusions; map TSS/TES.
Recommended services: Full-Length Transcript Sequencing, lncRNA Sequencing, TAIL Iso-Seq.
Direct RNA & RNA modification studies
Sequence native RNA without RT; investigate modification-associated signals.
Recommended service: Direct RNA Sequencing.
DNA methylation / epigenetics (5mC/6mA)
Call modifications from signal to build methylome maps and DMRs.
Recommended services: Standard/Ultra-Long WGS, Targeted (Cas9/Adaptive).
Target discovery & validation
Enrich loci of interest quickly without full-genome cost.
Recommended services: Targeted Nanopore Sequencing (Cas9 or Adaptive Sampling), Amplicon Sequencing.
3D genome architecture
Generate long-range contact maps for scaffolding and chromatin studies.
Recommended service: Pore-C.
Metagenomics & pathogen surveillance
Improve assembly/strain resolution; benefit from real-time decisions.
Recommended services: Standard WGS, Targeted, Amplicon (per design).
For high-depth small-variant cohorts, short-read can be cost-efficient; hybrid designs (short-read + Nanopore) capture both depth and long-range context.
Nanopore vs PacBio HiFi vs Illumina — Platform Comparison
Choosing the right platform for your project? Here is how Nanopore compares to PacBio HiFi and Illumina on the dimensions that matter for research outcomes.
| Dimension | Nanopore (ONT) | PacBio HiFi | Illumina |
| Principle | Ionic current through a protein nanopore; neural-network basecalling | Optical detection in ZMWs; circular consensus (CCS) | Sequencing-by-synthesis; cluster imaging |
| Read length (typical) | 10–100 kb routine; ultra-long to 2 Mb+ | ~15–20 kb HiFi reads; subreads up to ~100 kb | Up to 2×300 bp |
| Per-read accuracy (raw) | Q10–Q20 raw; improving with R10.4.1 + Dorado; high accuracy with consensus depth | QV ≥30 (≥99.9%) via CCS consensus | ≥99.9% raw |
| Data timing | Real-time streaming; stop or extend a run live | Batch (analysis after run completes) | Batch |
| Native biology | Direct RNA sequencing; 5mC/6mA from raw signal; RNA modifications without RT or amplification | 5mC from polymerase kinetics; no bisulfite needed | No native modification detection (standard workflows) |
| Where it shines | Ultra-long span (telomeres, massive SVs, gap closure); real-time/field work; Direct RNA | Highest long-read accuracy; assemblies and methylation from one dataset | Deep SNV/indel cohorts; cost-efficient large studies |
| Trade-offs | Raw accuracy lower than HiFi or Illumina; signal-aware bioinformatics required | Longer run times; no real-time control or streaming | No long-range context; cannot detect native modifications |
Quick decision guide:
- Need reads longer than 100 kb or field-deployable sequencing → Nanopore.
- Need the highest accuracy in long reads for assemblies and variant calling → PacBio HiFi.
- Need direct RNA sequencing or real-time data → Nanopore is the only option.
- Large SNV/indel cohorts on a budget → Illumina for depth; add Nanopore long reads for SVs and phasing.
- Hybrid designs: Nanopore ultra-long + PacBio HiFi for complete T2T assemblies; Nanopore long reads + Illumina short reads for SV detection with deep SNV validation.
Actual performance varies with sample quality, library preparation, sequencing depth, and analysis pipeline.
Quality & Study Design
- Replicates & Controls: Recommend ≥2 biological replicates per condition; include negatives for amplicon/targeted; optional spike-ins for methylation.
- Coverage Planning: Size long-read depth to genome complexity/SV goals; plan on-target depth for targeted/amplicon; size reads/sample for isoforms or Direct RNA.
- Acceptance Criteria: Pre-agreed targets for yield per barcode, on-target fraction, mapping rate, and read-length profile (e.g., N50 goals for Ultra-Long).
- Run-Time Decisions: Use real-time dashboards to stop/extend/reload efficiently; document any variances in the project memo.
The CD Genomics Advantage
Applications-first scoping
Map your biological question to the right Oxford Nanopore service (Ultra-Long, Direct RNA, Full-Length Transcript/lncRNA, Targeted/Cas9 or adaptive, Amplicon, Pore-C, TAIL Iso-Seq).
Design modeling & feasibility
Coverage modeling, barcode balance, and target/primer feasibility checks before you commit.
Reproducible analytics
Containerized/ version-locked pipelines; clean result packaging with analysis report and data dictionary.
Real-time run efficiency
Live dashboards to stop/extend/reload when goals are met; adaptive sampling when suitable.
Data integrity & security
Structured folders, checksum verification, and secured transfer with a stated retention policy.
Publication-grade support
Optional results walkthrough, figure/table preparation, and manuscript/review assistance.
Cross-platform neutrality
Objective guidance when hybrid strategies (short-read + ONT, or HiFi + ONT) add value.
Sample Requirements
| Service | Amount & Integrity | Purity | Storage / Shipping | Key Notes |
| WGS (Standard) | ≥1–3 μg gDNA, ≥30 kb | A260/280 1.8–2.0; A260/230 ≥2.0 | −20°C on ice | Provide extraction method; no vortexing |
| Ultra-Long WGS | ≥3–5 μg HMW gDNA, ≥50 kb preferred | A260/280 1.8–2.0; A260/230 ≥2.0 | −20°C | Gentle handling critical; wide-bore tips only |
| Direct RNA Sequencing | ≥500 ng–1 μg poly(A)+ RNA; RIN ≥7 | A260/280 ~2.0; A260/230 ≥2.0 | −80°C dry ice | Do not heat or denature before shipping |
| Full-Length Transcript | ≥500 ng–1 μg total RNA; RIN ≥8 | A260/280 ~2.0; A260/230 ≥2.0 | −80°C dry ice | Poly(A)+ or rRNA-depleted per design |
| Full Length LncRNA | ≥500 ng–1 μg total RNA; RIN ≥7 | Same as above | −80°C dry ice | rRNA depletion or poly(A)– selection |
| Amplicon Sequencing | ≥50–200 ng pooled amplicons | Clean PCR; no primer dimers | 4°C cold pack | Provide primer table and target gene |
| Targeted (Cas9/Adaptive) | ≥1–3 μg gDNA | A260/280 1.8–2.0 | −20°C | Provide target region BED file or gene list |
| Pore-C | ≥1–2 μg HMW gDNA, ≥50 kb | A260/280 1.8–2.0 | −20°C | Gentle handling; no vortexing |
| TAIL Iso-Seq | ≥500 ng–1 μg total RNA; RIN ≥7 | A260/280 ~2.0 | −80°C dry ice | Poly(A)+ selection required |
| Nano tRNA Sequencing | ≥500 ng total RNA; RIN ≥7 | A260/280 ~2.0; A260/230 ≥2.0 | −80°C dry ice | Provide tRNA enrichment method if self-enriched |
| Long Reads Amplicon | ≥100 ng pooled amplicons; ≥500 bp target | Clean PCR; no primer dimers | 4°C cold pack | Provide primer table + target coordinates |
General notes
- Avoid inhibitors (phenol, heparin, EDTA, polysaccharides) and repeated freeze–thaw; do not over-dry beads.
- Use wide-bore tips and no vortexing for HMW DNA.
- Include a brief extraction method and any known contaminants.
- Low-input/FFPE or unusual matrices may be feasible—contact us for a customized protocol.
Project Workflow
Client Case Study (Published Research)
Title: FIONA1-mediated methylation of the 3'UTR of FLC affects FLC transcript levels and flowering in Arabidopsis (Oxford Nanopore use case)
- Authors: Bin Sun, Kaushal Kumar Bhati, Peizhe Song, et al.
- Publication date: September 27, 2022
- Journal: PLOS Genetics
Research question (Attention):
Which enzyme installs m^6A at the 3′UTR of the FLOWERING LOCUS C (FLC) mRNA, and how does that modification affect FLC transcript stability and flowering?
Approach:
A multi-omics design integrated Oxford Nanopore Direct RNA sequencing, mRNA-seq, and meRIP-seq to profile differential expression and differential RNA methylation in wild type vs FIONA1 (FIO1) mutant plants. Direct RNA captured native signal features while meRIP-seq mapped m^6A-enriched regions; combined evidence pinpointed the modification site at the FLC 3′UTR.
Key findings:
- FIO1 is the methyltransferase responsible for 3′UTR m^6A on FLC mRNA.
- Loss of this 3′-end methylation in fio1 mutants destabilizes FLC mRNA (reduced FLC levels) and contributes to an early-flowering phenotype.
- The authors released Nanopore Direct RNA data (GEO: GSE212766) and matched mRNA/meRIP datasets, supporting reproducibility.
Direct RNA-sequencing analysis.
What this demonstrates:
This peer-reviewed study shows how Nanopore Direct RNA sequencing—paired with analysis of RNA modifications (m^6A)—answers biological questions that depend on native RNA and post-transcriptional regulation. It's a strong exemplar of "nanopore sequencing technology, bioinformatics, and applications" for epitranscriptomics and gene-regulatory mechanisms.
How CD Genomics would scope a similar project:
- Service: Nanopore Direct RNA Sequencing (+ optional meRIP-seq via partner workflow)
- Analysis: isoform profiling, modification-associated signal summaries, differential expression, integration with immunoprecipitation-based m^6A peaks
- Deliverables: FASTQ (Direct RNA), optional POD5/FAST5, QC report, modification summaries, figures/tables suitable for publication
FAQs — Nanopore Sequencing
FIONA1-mediated methylation of the 3’UTR of FLC affects FLC transcript levels and flowering in Arabidopsis
PLoS Genetics | 2022Complete Genome Sequence of the Lignocellulose-Degrading Actinomycete Streptomyces albus CAS922
Microbiology Resource Announcements | 2020The m6A writer FIONA1 methylates the 3’UTR of FLC and controls flowering in Arabidopsis
bioRxiv | 2022
