Oxford Nanopore Technology
User Guide
The instructions in this guide must be followed carefully to achieve the best possible results and avoid delays in processing your request.
Samples that do not comply with the recommendations may be rejected without compensation.
- Starting Material
- Requirements – Quality and Quantity
- Required Containers and Sample Identification
- Service Request Form and Sample Submission
- Sample Shipment Preparation
- Transmission of results
Starting Material
| Plasmid | PCR product |
|---|---|
| Plasmid DNA circular, double-stranded and less than 25kb. | PCR product more than 1500 bp and less than 25 kb. |
| – Recommended extraction kit: QIAGEN kits – Homemade protocol: an additional purification step with a commercial kit is required. Otherwise, sequencing quality may be compromised or fail. | – PCR products must have been purified by a magnetic bead-based method such as AMPure*. *Bead purification service is offered for an additional fee and processing time. Contact Client Management Office for more information. |
Requirements – Quality and Quantity
Samples must be eluted in 10 mM Tris buffer, pH 8.5, or nuclease-free ultrapure water.
| Sample type | Volume | Concentration | Quality criteria to be met | Recommended verification method | Impact on sequencing |
|---|---|---|---|---|---|
| Plasmid | 20 µL | 40 ng/µL to 80 ng/µL | Circular, double-stranded, intact | Size and integrity verified by electrophoresis gel | Degraded/fragmented sample: May lead to sequencing failure resulting in lack of consensus due to absence of full reads |
| Purified PCR product | 20 µL | 40 ng/µL to 80 ng/µL | Linear, double-stranded, intact | ||
| Unpurified PCR product (extra service)* | 20 µL | 60 ng/µL to 120 ng/µL |
Low-concentrated samples are often degraded, which significantly affects the quality of the results.
Overly concentrated samples will result in sequencing failure.
It is imperative to dilute the samples to the required concentration or a dilution fee will be charged.
Concentration Verification
- Recommended method: Qubit or equivalent fluorometric method.
Note: Spectrophotometric methods such as Nanodrop are not recommended for measuring DNA concentration because they tend to overestimate the DNA concentration, particularly for low-concentration samples or those obtained from complex extractions.
Purety Verification
- Recommended method: Nanodrop or other spectrophotometric method.
| Optical density ratio | Expected value | Problems related to out-of-range values | Impact on sequencing |
|---|---|---|---|
| 260/280 | Between 1.8 and 2.0 | Contamination by proteins or phenol. Or an unstable measurement due to an insufficient concentration. | Poor sequencing yield or failure |
| 260/230 | Between 2.0 and 2.2. | Contamination by salts, solvents, or chemical residues. Or an unstable measurement due to an insufficient concentration. | Poor sequencing yield or failure |
- If using a Qiagen kit: perform the optional PB buffer wash step to increase purity.
Required Containers and Sample Identification
- 16 samples or less: 1.5mL tube
- 17 samples or more: 96-well PCR plate
Tube and plate identification:
- Unique, short, and legible name.
- Identical to the name indicated on the submission form.
- Plate samples – Column arrangement required, as indicated on the form.
This is to avoid any delays in processing the application.
| Required tubes | 96-well PCR plate recommended | 96-well PCR plate unaccepted |
|---|---|---|
| – 1.5mL tubes All types of 1.5mL tubes. are accepted. | – Half-skirt PCR plate All types of clear half-skirt clear 96-well PCR plates are accepted. Example: Thermo-Fast 96 PCR detection plate with flat deck; Life Technologies, Cat# AB1400L – Full-skirt PCR plate All types of clear full-skirt clear 96-well PCR plates are accepted. Example: Microseal PCR plates 96-well clear; Bio-Rad, Cat# MSP9601 | – 96-well cell culture plates – Opaque 96-well PCR plates |
| Plates should be sealed with 8 or 12-well strip caps or heat seals to ensure a tight seal of each well. Adhesive sealants should be avoided due to the high risk of leakage and cross-contamination. |
Service Request Form and Sample Submission
Click on service request and sample submission to view instructions.
It is highly recommended to provide a reference sequence for PCR product sequencing. This is to facilitate consensus sequence reconstruction if the amplicon quality is poor, especially if the sample contains more than one amplified sequence.
The only accepted formats are Fasta (.fa, .fasta), SnapGene (.dna), Genbank (.gb, .gbk), Ape (.ape), text (.txt, sequence only, without header or other information).
Sample Shipment Preparation
Click on samples shipment preparation to view instructions.
Transmission of Results
An automated message from Nanuq is sent to the submitter as soon as the sequences are available.
Sequencing results are directly accessible via the Nanuq web application.
Each sequencing result folder contains:
| Plasmid | PCR product |
| • A consensus sequence (.fasta). • A file with statistics for each base of the consensus sequence (.xlsx). • Two graphs reporting the quality and size of the reads observed during sequencing (.png). • An annotated map of the consensus sequence that can be viewed in a web browser (.html) or with software such as snapgene or snapgene-viewer (.gbk). • An alignment of the consensus sequence with the reference sequence (if provided) (.aln). This alignment can be conveniently viewed using snapgene or snapgene-viewer software. | • A consensus sequence (.fastaq). • A file with statistics for each base in the consensus sequence (.xlsx). • A sequencing report that can be viewed in a web browser. |
These files can be downloaded locally.
All samples will be retained for a maximum of 2 weeks after sequencing before being destroyed.
Version 1.0_2025-10-01