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
| Starting Material |
|---|
| Bacterial genomic DNA. |
| – Recommended extraction kit: commercial kits from NEB, QIAGEN, or Zymo. – In-house protocol: an additional purification step with a commercial kit is required. If these recommendations are not followed, the quality of the sequencing may be compromised, or even result in failure. |
Requirements – Quality and Quantity
The quantity and quality of samples are crucial for obtaining quality sequences.
| Sample Type | Quantity | Concentration | Volume | Quality criteria to be met | Recommended verification method | Impact on sequencing |
|---|---|---|---|---|---|---|
| Bacterial genomic DNA | 3 µg | ~ 100 ng/µL ideally Low-concentration samples produce shorter readings and generate lower sequencing yields. | 20 µL minimum 200 µL maximum | 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 |
Samples must be eluted in 10 mM Tris buffer, pH 8.5, or nuclease-free ultrapure water
The client is responsible for providing samples of good quality and in sufficient quantity. Sequencing failures due to non-compliance with requirements will be billed.
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.
Purity Verification
Recommended method: Nanodrop or other spectrophotometric method.
| Ratio | Expected value | Problem if out of bounds | 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 request.
| Required tubes | 96-well PCR plate recommended | 96-well PCR plate not accepted |
|---|---|---|
| – 1.5mL tubes All types of 1.5mL tubes are accepted. | – Half-skirt PCR plate All types of clear half-skirt 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 |
| Sealing Required: 8 or 12 strip caps or heat sealing To be avoided: adhesive sealants (risk of leakage or contamination) |
Service Request Form and Sample Submission
Click on service request and sample submission to view instructions.
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:
| Bacterial genome |
|---|
| – A complete report with statistics and information for each sample – Raw FASTQ files – An assembly (.fasta) – An annotation file (.gff) – An annotated assembly in GenBank format (.gbk) |
These files can be downloaded locally.
All samples will be retained for a maximum of 2 weeks after sequencing before being destroyed.