Sample Submission Guidelines
Why Ultra-Long Nanopore Sequencing Matters
Conventional sequencing often leaves unresolved gaps, particularly in genomes with extensive repeats, polyploidy, or high structural complexity. These gaps limit the accuracy of genome assemblies and reduce confidence in downstream analyses.
Nanopore ultra long sequencing addresses these limitations by generating DNA reads far longer than standard approaches. Unlike short- or mid-length sequencing, which struggles with highly repetitive regions, ultra long reads can span centromeres, telomeres, and structural variants in a single stretch.
This capability has transformed genome research. Plant and animal studies now routinely achieve telomere-to-telomere (T2T) assemblies, enabling researchers to explore genetic architecture with unprecedented resolution. For applied research in crop breeding, biomedical discovery, and microbial evolution, the ability to resolve complete genomes provides a direct competitive advantage.
Technical Parameters
| Parameter | Specification |
|---|---|
| Read length | N50 >50–100 kb; maximum reads >4 Mb |
| Input requirement | ≥6 million cells (PBMCs, cultured cells, or frozen tissue) |
| Chemistry | Nanopore Ultra-Long Sequencing Kit (SQK-ULK114, Kit 14, R10.4.1 nanopore) |
| Platform | PromethION / GridION |
| Throughput | Up to 90–100 Gb per PromethION flow cell |
| Accuracy | Raw read Q20+ (Kit 14 chemistry) |
| Preparation time | ~200 minutes plus overnight elution |
| QC methods | Qubit, Nanodrop, pulsed-field gel electrophoresis (PFGE) |
| Storage & logistics | Kits shipped at 2–8 °C; long-term storage at –20 °C |
Advantage: CD Genomics Service Highlights
CD Genomics delivers an end-to-end nanopore ultra long sequencing service that combines advanced laboratory protocols with proven sequencing platforms. Our approach is designed to maximise read length, stability, and accuracy, enabling researchers to close gaps and produce highly contiguous assemblies.
Key Service Advantages
- Gapless assemblies: Ultra long DNA sequencing nanopore spans repetitive and GC-rich regions, eliminating assembly gaps.
- Structural variant discovery: Long reads detect large insertions, deletions, inversions, and repeat expansions with confidence.
- Polyploid genome resolution: Optimised strategies validate haplotype phasing in complex species.
- Telomere-to-telomere analysis: Nanopore long read sequencing supports complete T2T assemblies in plants and animals.
- Optimised chemistry: Use of the Nanopore Ultra-Long Sequencing Kit (SQK-ULK114) with Kit 14 ensures high yield and Q20+ raw read quality.
- Flexible applications: Compatible with genomic DNA and integrated with downstream nanopore RNA sequencing solutions.
- Proprietary SOPs for extracting ultra-high molecular weight DNA from plants, animals, and microbial samples.
- Workflow improvements in DNA repair and library construction that preserve fragment length >100 kb.
- Access to PromethION and GridION platforms with the latest Kit 14 chemistry for Q20+ accuracy.
Proven Case Evidence
| Project | Strategy / Data Output | Key Results |
|---|---|---|
| Wheat genome assembly | HiFi + ONT ultra long reads | Contig N50 improved from 341 kb to 2.15 Mb; near-gapless reference achieved |
| Sugarcane genome validation | Ultra long nanopore reads for haplotype verification | Switch error rate as low as 0.05/Mb; >90% mapping accuracy |
| Chili pepper T2T genome | Four PromethION flow cells; N50 read length up to 107 kb | Average N50 = 91.5 kb; longest read 2.98 Mb; complete T2T assembly |
| Sorghum genome assembly | ONT ultra long sequencing only (no Illumina/PacBio) | Completed telomere-to-telomere assembly; validated centromeres and telomeres |
Key Applications
De novo genome assembly
Ultra long reads span repetitive elements and GC-rich regions, closing gaps that remain unresolved with short-read or standard long-read sequencing.
Structural variation detection
Nanopore long read sequencing identifies large insertions, deletions, inversions, and repeat expansions that influence genome stability and phenotype.
Polyploid genome analysis
Ultra long DNA sequencing nanopore improves haplotype phasing and validates assemblies in polyploid plants and hybrid species.
Telomere-to-telomere (T2T) assembly
Complete chromosome-level assemblies are achieved by spanning telomeres, centromeres, and rDNA regions with single reads.
Transcriptome and RNA sequencing integration
Combined with Nanopore Direct RNA Sequencing and Nanopore Full-Length Transcript Sequencing, researchers can link genome structure with transcriptional activity.
Workflow: End-to-End Service
CD Genomics offers a complete workflow for nanopore ultra long sequencing, from sample preparation to final data delivery. Each stage is optimised to preserve ultra-high molecular weight DNA and maximise read length.
1. Sample preparation
- Support for plant, animal, and microbial samples
- Specialised protocols to minimise degradation and remove polysaccharides or secondary metabolites
2. DNA extraction and QC
- Extraction of ultra-high molecular weight (uHMW) DNA
- Quality checks using Qubit, Nanodrop, and pulsed-field gel electrophoresis
3. Library preparation
- Nanopore Ultra-Long Sequencing Kit (SQK-ULK114) with Kit 14 chemistry
- Transposase-based fragmentation and rapid adapter ligation
- Overnight elution to preserve long DNA molecules
4. Sequencing
- Performed on PromethION or GridION platforms
- Achieve read lengths >100 kb N50, with maximum reads exceeding 4 Mb
5. Bioinformatics analysis
- Real-time basecalling and quality filtering
- Assembly, polishing, and variant detection
- Optional telomere-to-telomere (T2T) assembly support
6. Reporting and delivery
- FASTQ and BAM files with QC metrics
- Customised analysis reports for genome assembly or structural variant studies
Bioinformatics Analysis
Basic Analysis
| Analysis Stage | Description |
|---|---|
| Basecalling | Converts raw electrical signals into DNA/RNA sequences using models like Dorado for improved accuracy. |
| Quality Control (QC) | Includes read length distribution, coverage metrics, and quality scores. |
| De novo Assembly | Builds high-contiguity genomic assemblies using tools optimized for long reads (e.g., Flye, Canu) . |
| Polishing & Error Correction | Refines assembly error profiles using long-read self-correction or hybrid polishing workflows . |
Advanced Analysis
| Analysis Stage | Description |
|---|---|
| Structural Variant Calling | Detects large indels, duplications, inversions using tools such as Sniffles or CuteSV. |
| Variant Phasing & SV Annotation | Phases variants across long contigs and annotates SVs for biological interpretation. |
| Telomere-to-Telomere (T2T) Support | Completes chromosome-level assemblies by closing gaps at repeats, centromeres, and telomeres. |
| Epigenetic & Base Modification Detection | Detects DNA/RNA modifications (e.g., 5mC, m6A) using Remora or Megalodon during basecalling. |
| Metagenomic / Taxonomic Classification | Classifies reads in mixed samples using workflows like EPI2ME meta pipelines. |
Deliverables
Clients will receive:
- Raw sequencing data (FASTQ format)
- Quality control report (read length distribution, N50, yield, accuracy)
- Optional genome assembly and variant analysis results
- Project summary report
Sample Requirements for Nanopore Ultra-Long Sequencing
| Category | Requirements |
|---|---|
| Sample type | High-quality genomic DNA (animal tissue, plant tissue, microbial culture, or blood cells) |
| Buffer | DNase-free water, Elution Buffer, or 10 mM Tris pH 8.0 |
| Purity | OD260/280 ratio 1.8–2.0; OD260/230 ≥ 2.0; DNA must be RNase-treated, free of protein, polysaccharide, or phenolic contamination |
| Integrity | Ultra-high molecular weight DNA; minimal shearing; confirm by PFGE or TapeStation |
| Concentration | ≥20 ng/µL (Qubit measurement recommended; if using Nanodrop, double the concentration due to overestimation) |
| Total amount | ≥5 µg genomic DNA per sample (recommended ≥10–15 µg for ultra-long read workflows) |
| Volume | ≥200 µL in DNA LoBind tubes (1.5 mL or 2 mL centrifuge tubes preferred, sealed with parafilm for transport) |
| Sample labeling | Label top of each tube with ≤4 alphanumeric characters (e.g., A101); ensure names match submission form |
| Shipping | Transport on dry ice; protect tubes inside 50 mL tubes or rigid containers with cushioning to prevent breakage |
| Avoid | Repeated freeze–thaw cycles, EDTA-containing buffers, degraded DNA, or contamination from secondary metabolites |
Demo Results Showcase
Read length distribution
The accompanying chart (from Oxford Nanopore Technologies) illustrates how ultra-long sequencing dramatically extends read length compared to standard ligation methods—producing a smooth tail reaching several hundred kilobases.
Wheat genome impact
Incorporating ultra-long ONT reads increased the contig N50 from 341 kb to 2.15 Mb, leading to a near-gapless assembly ideal for downstream genomic research and breeding programs.
Plant T2T advancement
In recent plant genome projects, optimized extraction and library protocols delivered ultra-long reads with an N50 up to 440 kb, significantly enhancing automated telomere-to-telomere (T2T) assembly.
Sorghum assembly success
A complete T2T sorghum genome was assembled using only ONT ultra-long sequencing, demonstrating the method's capability to resolve full chromosomes without needing complementary technologies.
FAQ (Frequently Asked Questions)
Q: What read length can I expect from nanopore ultra-long sequencing?
You can expect N50 read lengths typically ranging from 50 to over 100 kb, and in optimal conditions individual reads can exceed 4 Mb—ultra-long reads enable resolution of complex genomic regions like repeats and centromeres.
Q: Why does ultra-long read length matter for my genome assembly?
Ultra-long reads span highly repetitive or structurally complex regions, enabling gapless or near gapless assemblies such as T2T genomes and improving detection of structural variants that shorter reads cannot resolve .
Q: What impact do long reads have on structural variant detection?
Long nanopore reads improve the discovery of large insertions, deletions, inversions, and repeat expansions by spanning them directly, which simplifies variant calling and reduces ambiguity.
Q: Can nanopore sequencing handle both DNA and RNA samples?
Yes, nanopore sequencing supports direct sequencing of both DNA and RNA molecules without the need for amplification or labelling, which allows you to study transcripts and base modifications alongside genome structure.
Q: What factors affect sample quality for ultra-long reads?
DNA purity and fragment length are critical. High-quality extraction with minimal degradation is essential, and multiple extraction attempts may be needed to obtain ultra-high molecular weight DNA suitable for ultra-long read sequencing.
Q: Is nanopore technology suitable for field or portable applications?
Yes, because nanopore sequencers can process native DNA or RNA in real time in scalable formats—from portable MinION devices to high-throughput platforms—this flexibility supports lab, field, and remote applications
Case Study: Human Genome Assembly with Nanopore Ultra-Long Reads
1. Background
The human genome is ~3.1 Gb in size and contains extensive repetitive regions, segmental duplications, and heterozygosity, making it challenging to assemble using short-read sequencing. Conventional technologies fail to resolve centromeres, telomeres, and structural variants, leaving persistent gaps in reference genomes. This case study explores how nanopore ultra long sequencing can overcome these barriers.
2. Methods
Researchers sequenced the GM12878 human cell line (Utah/CEPH pedigree) on the Oxford Nanopore MinION platform with R9.4 1D chemistry. DNA preparation protocols were designed to minimise shearing and preserve ultra-high molecular weight fragments.
- Data generated: 91.2 Gb of sequence (~30× coverage) from 39 flow cells
- Ultra-long reads: N50 >100 kb; maximum read length 882 kb
- Assembly tool: Canu assembler with Illumina short-read polishing for accuracy improvement.
3. Results
- Nanopore-only assembly NG50 = ~3 Mb
- Adding 5× ultra-long coverage doubled NG50 to 6.4 Mb
- Major histocompatibility complex (MHC) (4 Mb) resolved in a single contig
- 12 large gaps (>50 kb) in GRCh38 were closed
- Final accuracy after polishing = 99.88%
- Ultra-long reads enabled haplotype phasing across the entire MHC locus.
Chromosome plot illustrating how nanopore ultra long sequencing closed 12 gaps in GRCh38, including the 16 Mb MHC locus. Continuous colour blocks represent contiguous assembly, while white gaps indicate unresolved regions.
4. Conclusions
This study demonstrates that nanopore ultra long read sequencing enables highly contiguous human genome assemblies. The technology resolved complex loci, closed reference gaps, and provided haplotype phasing at chromosome scale. These results highlight its potential for producing near-complete telomere-to-telomere (T2T) assemblies and advancing both fundamental genomics and translational applications.
References:
- Lu D, Liu C, Ji W, Xia R, Li S, Liu Y, Liu N, Liu Y, Deng XW, Li B. Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-to-telomere genome assembly. Mol Plant. 2024 Nov 4;17(11):1773-1786. doi: 10.1016/j.molp.2024.10.008. Epub 2024 Oct 16. PMID: 39420560.
- Prall, T.M., Neumann, E.K., Karl, J.A. et al. Consistent ultra-long DNA sequencing with automated slow pipetting. BMC Genomics 22, 182 (2021).
- Jain M, Koren S, Miga KH, Quick J, Rand AC, Sasani TA, Tyson JR, Beggs AD, Dilthey AT, Fiddes IT, Malla S, Marriott H, Nieto T, O'Grady J, Olsen HE, Pedersen BS, Rhie A, Richardson H, Quinlan AR, Snutch TP, Tee L, Paten B, Phillippy AM, Simpson JT, Loman NJ, Loose M. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol. 2018 Apr;36(4):338-345. doi: 10.1038/nbt.4060. Epub 2018 Jan 29. PMID: 29431738; PMCID: PMC5889714.
