User Guide
The instructions in this guide must be followed carefully to achieve the best possible results and avoid delays in processing your request.
Any libraries that do not comply with the recommendations may be rejected without compensation.
Note that delays in the processing of libraries will vary depending on the size of the project. It is recommended to contact the Client Management Office for information regarding processing time.
- General Information
- Starting material
- Requirements – Volume and concentration
- Required Containers and Sample Identification
- Service Request Form and Sample Submission
- Sample Shipment Preparation
- Transmission of results
General Information
Library structure for Illumina sequencing
Library preparation for Illumina sequencing can be performed using commercial kits or customized protocols with specific primers.
A sequencing-ready library has adapters ligated to both ends of the DNA insert. These adapters contain:
- P5 and P7 sequences: enable binding to the flow cell.
- Sequencing primers: used for Read 1, Read 2, and index reads.
- Indexes (i5 and i7): short sequences (6–10 bases) used to distinguish samples during multiplexing. Index 1 (i7) is read first, followed by Index 2 (i5).
- Unique Molecular Identifiers (UMIs): sequences of 10–18 bases that allow identification of each DNA fragment and differentiation of PCR duplicates. Their position varies depending on the kit used (after i7, replacing i5, or at the beginning of Read 1).
Inline barcodes (integrated directly into Read 1 or 2) should not be confused with Illumina indexes. They are not supported by standard pipelines (including Nanuq, the CES web application).
Figure 1 – Structure of an Illumina library
Diagram showing the construction of a sequencing-ready library. Adapters ligated on both sides of the insert contain the sequences required for binding to the flow cell (P5 and P7), sequencing primers (Read 1, Read 2, Index 1, Index 2), and indexes (i5 and i7) used for multiplexing. Depending on the kit used, a molecular index (UMI) may also be present.
Adapter sequence references
The exact Illumina adapter and primer sequences are not reproduced in this guide. They are available on Illumina’s official website: Illumina Adapter Sequences
Library sequence complexity
Illumina technology requires sufficient base diversity, particularly at the beginning of the read, to ensure optimal data quality.
Libraries that do not exhibit a random distribution of bases (low complexity) must therefore be clearly identified, as they can reduce sequencing performance.
Such libraries include:
• Amplicons (PCR)
• RAD libraries (restriction site–associated DNA)
• Reduced-complexity libraries used for methylation analysis, such as those generated by bisulfite conversion or enzymatic methylation (e.g., MethylSeq / EM-seq)
To compensate for low complexity, the sequencing facility may add a control library (PhiX174, Illumina) at a level of 10 to 50%, depending on the initial library. However, this addition decreases the proportion of reads assigned to the samples of interest.
Sequencing data will be provided as is. The CES cannot be held responsible for issues related to the design, quality or sequence complexity of the libraries.
Diversity of indexed libraries and multiplexing recommendations
To ensure adequate diversity during index reads, it is recommended to pool at least three indexed libraries per sequencing lane. This practice helps maintain data quality, even for libraries with normal complexity.
Specific requests regarding library pooling cannot always be accommodated.
The CES reserves the right to modify multiplexing schemes without prior notice. Any special request must be indicated in the Comments column when filling the Sample Submission Sheet.
Starting material
The starting material is a library or a pool of libraries that carries adaptors for Illumina sequencing.
Requirements – Volume and concentration
- The libraries should be diluted in Tris-HCl (10 mM) buffer or in nuclease-free water.
- The libraries must not contain chelating agent (ex. EDTA) or the concentration must be reduced to 0.1mM.
| Requirements | Minimum | Maximum |
|---|---|---|
| Volume | ≥ 25 µL per library* | 110 µL |
| Concentration | ≥ 2 nM for each library* | 60 ng/µL |
- The volume entered in the sample submission form must exactly match the physical volume in the plate.
- Volume exceeding 110 µL will be discarded without notice
- Should the library volume be inferior to the minimum volume, it will be diluted to an appropriate volume without notice.
- Library concentration must not exceed 60 ng/µL. Over-concentrated libraries will be diluted; QC will be repeated and may cause additional fees and delays.
- Replacement libraries must meet total requirements. Partial replacement (« top-ups») will not be accepted.
- It is recommended to send an aliquot of your library. Sequencing-ready libraries will not be returned and will be discarded 3 months after the completion of the project without notice.
Concentration verification
Recommended method: quantification by qPCR, which specifically targets the adapters required for Illumina sequencing.
Note: UV absorbance–based methods (e.g., NanoDrop) or fluorescence-based methods (e.g., PicoGreen) measure total DNA but cannot distinguish molecules carrying the P5 and P7 adapters.
Sequencing primers
Custom sequencing primers (if required) should be sent in 1.5 mL tubes.
Tubes must be properly identified:
- Principal Investigator last name and primer name
- Primer concentration
- Sequencing read (Read 1, Read 2, Index 1 or Index 2)
| Requirements | Volume | Concentration |
|---|---|---|
| NovaSeq | ≥ 30 µL * *minimum volume per primer and per sequencing lane | 100 µM |
| NextSeq | N/A | N/A |
Required Containers and Sample Identification
- All library shipments must be made in 96-well PCR plates.
- Each 96-well plate must correspond to a single submission form and be labeled exactly as indicated.
- Libraries must be arranged in the plate according to the layout specified in the sample submission form.
| Required 96-well plates | Unaccepted 96-well plates |
| Full-skirt PCR plates – BioRad Hard-Shell 96-Well PCR Plates, skirted, Cat# HSP9601 – Eppendorf twin.tec, Cat# 951020401 – Corning Thermowell GOLD, Cat# 3752 – Axygen 96-well PCR Microplate, Cat# PCR96FSC – 96 well 0.2mL Skirted PCR plate – Grey ultra rigid frame, Cat# IST-601-096GCT | – 96-well cell culture plates – No-skirt 96-well PCR plates – Half-skirt 96-well PCR plates |
| Recommended adhesive films – Clear adhesive film Life Technologies MicroAmp® Clear Adhesive Film, Cat# 4306311 – Aluminum adhesive films VWR Aluminum Foils for PCR and Cold Storage, Cat# 60941-074 |
Service Request Form and Sample Submission
Click on request a service and submit samples to view the instructions.
Contact the Client Management Office for any questions regarding the submission form, particularly concerning adapters, indexes, and whether custom indexes were used for library construction.
Sample Shipment Preparation
Click here for instructions on preparing samples for shipment.
Transmission of results
An automated notification from Nanuq will be sent to the principal investigator and project contacts once sequencing data are available.
Sequencing results are directly accessible via the Nanuq web application.
For more information on downloading data and available files, please refer to the Frequently Asked Questions page.