Technology Overview
Oxford Nanopore sequencing is an advanced third generation sequencing platform that offers high throughput sequencing of DNA or RNA samples, ranging from short to ultra-long reads. It operates by utilizing nanopores to capture library molecules and measuring the disruption of electric current, which accurately translates into DNA or RNA sequences. This cutting-edge technology enables the generation of long reads, facilitating the assembly of high-quality de novo genomes. Additionally, a barcoding system is available, allowing for multiplexed sequencing on a flow cell, thus achieving enhanced efficiency in terms of time and cost.
Nanopore Sequencing Services
| Sample Type | Service Type | Feature |
| DNA | Whole Genome Sequencing | • Long read lengths • 10kb & >25kb protocol available |
Service Charges and Ordering
Service Charges
The Core offers comprehensive services including nucleic acid extraction, library preparation, Nanopore sequencing and Bioinformatics services. Please contact Dr Hin Kwok (hinkwok.cpos@hku.hk / 2831-5483) or platform specialists for project discussion and official service quotation.
Price varies with the following factors:
- Throughput or fold coverage required (sequencing reads or bases output)
- Type of work (Microbial genome sequencing, targeted DNA sequencing, RNA-seq, metagenomics etc.)
- Number of samples
- Method for library preparation
- Bioinformatics analysis needs
- Special and/or additional requirements
- Affiliated institute (HKU, local academics, oversea academics and commercial company)
Service Ordering
Please submit service request through iLab.
Technical Details
Library Preparation
Different library preparation options are available depending on the application. We will be happy to discuss your needs and provide recommendations.
Sequencing Run
MinION can deliver up to 20 Gb data in 1 flow cell.
PromethION can deliver up to 100 Gb data in 1 flow cell. The system also provides flexibility in run size, allowing up to 24 flow cells per run. Different library preparation options are available.
* Performance will vary based on sample quality and other experimental factors.
Workflow
Sample Requirement and Submission
A. Sample Preparation
Pure DNA is crucial to ensure consistent library preparation and quality data.
Our recommendations:
- Purification is required on samples that have undergone phenol/chloroform extraction methodology
- Treat DNA with RNase
Generally, samples should be:
- Free of inhibitors, e.g. heme (from blood), EDTA and salts
- Free of organic solvents, e.g. phenol, chloroform and ethanol
- Free of cellular debris or proteins
- Free from original organism/tissue (haem, humic acid, polyphenols, etc.)
- Dissolved in buffer at proper pH, e.g. 10mM Tris-HCl at pH 8.5
- Not degraded, i.e. high-molecular weight, double-stranded genomic DNA and RNA with high RIN score on bioanalyzer
B. Sample Requirements
DNA shall be assessed by UV spectrophotometry for purity and quantity estimation before sample submission. Gel photos are also required for some of the DNA-based applications.
After sample submission, sample QC will be performed by the Core platform specialists, which includes Qubit quantification for DNA samples.
| Application | Protocol option | Sample requirements |
| Whole Genome Sequencing | 10kb protocol | 5 μg gDNA (Qubit measurement) OD 260/280 = 1.8 OD 260/230 = 2.0–2.2 DNA concentration: >50 ng/μL Volume: > 50 μL Fragment size: >20 kb preferred |
| >25kb protocol | 25 μg gDNA (Qubit measurement) OD 260/280 = 1.8 OD 260/230 = 2.0–2.2 DNA concentration: >100 ng/μL Volume: > 50 μL Fragment size: >30 kb preferred | |
C. Sample Submission
Please submit a service request through iLab to obtain project price and confirm the service content before sample submission. Please contact our colleagues in advance (via phone/email shown at the bottom of the webpage) before bringing the samples to the Core. With your samples, please bring along the following items:
- Hardcopy of completed NP Sample Submission Form, and the softcopy as attachment in iLab
- For DNA samples, gel photo with appropriate size marker, which should have an upper marker greater than or equal to 20 kb (e.g. GeneRuler 1 kb Plus DNA Ladder)
Please consult platform specialists before submission.
Data Collection
Once data is available, users will be notified via email. Details of data collection, please refer to CPOS Bioinformatics Core page.
Terms of Service
Data Throughput
Please refer to Technical Details for the information.
The actual throughput, however, will vary with the following factors:
- Sample quality
- Library preparation method
- Reagent lots
- Other run-to-run variations
Data Quality and Availability
We will in good faith produce the best output and quality data to all collaborators and users.
Any issue will be discussed openly before, during and after the run.
Data at various steps are also available to share with collaborators and users upon request.
Turnaround Time
Time for completion of service may vary depending on the number of samples submitted and the level of service demand at sample submission.
Samples will be put in a processing queue as soon as the samples and reagents are all ready. It takes ~1 month for reagent delivery from Oxford Nanopore Technologies.
Estimated completion date will be informed at time of sample submission.
Useful Link
For further information
Contact
Dr KWOK, Hin
Mr CHOW, Clement
Ms KONG, Carol
