Sequencing
CTG provides service for sequencing of user-prepared libraries on NovaSeq X Plus and NextSeq 2000.
Important! Sequencing Only project only includes time on the requested sequencer, requested sequencing reagents and hands-on time from CTG staff for operating the instrument. Preparation of the sequencing pool is your responsibility!
After data delivery, data will be kept by CTG for 1 month and subsequently deleted. We strongly recommend you to backup your data, i.e. have at least two copies. CTG cannot help you if your data gets corrupt or lost!
As we are using Illumina sequencing platforms, we provide some general information below. However, as the libraries are not prepared at CTG for these types of projects we cannot guarantee the output of the sequencing run. For the sequencing recommendations, we refer you to the library preparation kit manufacturer. If you have more specific questions regarding preparing the library pools for loading on the requested sequencer, please contact Illumina tech support.
General information before starting a Sequencing Only project
Please read through the following steps:
- Your libraries need to be compatible with the requested Illumina platform.
- NovaSeq libraries need to have unique dual indexes.
Read about unique dual indexes here.
- Pools can only be run on whole flow cells.
- Define a sequencing kit, number of cycles and loading concentration.
- Optimal pool loading concentration depends on the library type, insert size and is specific to the sequencing machine and kit.
- Illumina has guidelines about library loading concentration, but these often need optimization.
- If library preparation kit is not manufactured by Illumina, please check with the kit producer.
- CTG may give you guidance but cannot take responsibility for the optimization.
- You will be asked to provide a SampleSheet compatible with the requested sequencer that will allow demultiplexing of the sequencing data (FASTQ files).
- A template will be provided in iLab upon initiating a project request.
Library concentration and quality check
We recommend you do the following steps before and after pooling your libraries:
- Verify concentration of libraries using a fluorometric assay.
- Perform quality and size calculation of libraries on a micro-capillary gel electrophoresis system e.g. BioAnalyzer, TapeStation etc..
- Make sure no traces of primer dimers etc. remain.
- Calculate molarity of libraries based on the concentration and library size.
- Dilute and pool your libraries in RSB buffer to the final concentration.
- Verify concentration of the pool using a fluorometric assay before delivery.
- Calculate molarity of the diluted pool using pool concentration and average library size.
Requesting a project at CTG
To initiate a project using our available services, please start a project in iLab. You can login using your LucatID (SWAMID) on the Lund University iLab webpage
NovaSeq X Plus
| Pool volume | Flowcells come with 8 lanes. 10B flowcell: 100 µl (minimum 50 µl) per lane 25B flowcell: 140 µl (minimum 70 µl) per lane |
| Pool molarity | The optimal loading concentration needs to be optimized empirically and through guidelines from the kit manufacturer. Every library behaves differently and there is no linear correlation between loading concentration and cluster density. CTG may give you guidance but cannot take responsibility for this optimization. |
| Indexes | For demultiplexing (FASTQ files) you must supply Illumina compatible SampleSheet. A template will be provided in iLab upon initiating a project request. Please refer to the index manufacturer´s documentation for the correct index sequences. |
NextSeq 2000
| Pool volume | 60 µl (minimum 30 µl) |
| Pool molarity | The optimal loading concentration needs to be optimized empirically and through guidelines from the kit manufacturer. Every library behaves differently and there is no linear correlation between loading concentration and cluster density. CTG may give you guidance but cannot take responsibility for this optimization. |
| Indexes | For demultiplexing (FASTQ files) you must supply Illumina compatible SampleSheet. A template will be provided in iLab upon initiating a project request. Please refer to the index manufacturer´s documentation for the correct index sequences. |
When submitting a library for sequencing, you must provide a sample sheet (a .csv file) that specifies the names of the samples in a library pool and their indexes. After sequencing, a sample sheet is used to convert basecall files into .fastq.gz files.
Please fill in your information using the Illumina sample sheet template (available in iLab) to ensure correct formatting.
1. Index Orientation
The orientation of the i5 indexes in the sample sheet should be forward orientation for NextSeq 2000 and NovaSeq X Plus. The software will automatically reverse-complement the i5 indexes during sequencing.
2. Sample Sheet Fields
Your sample sheet must include the following details:
- Sample_ID: Name of each sample in the pool
- Index: i7 index sequences (forward orientation)
- Index2: i5 index sequences (forward orientation)
- Sample_Project: Project number in iLab for demultiplexed .fastq.gz files
3. Lane Information for Sample Sheets
When generating a sample sheet, it is important to structure lane assignments correctly to ensure proper sequencing.
A. If You Have Different Pools in Different Lanes:
Each pool should be assigned to a specific lane. For example:
- Pool 1 (3 samples) in Lane 1:
- Row 1: Lane 1, Sample 1
- Row 2: Lane 1, Sample 2
- Row 3: Lane 1, Sample 3
- Pool 2 (3 samples) in Lane 2:
- Row 4: Lane 2, Sample 1
- Row 5: Lane 2, Sample 2
- Row 6: Lane 2, Sample 3
B. If You Have One Pool Spanning All 8 Lanes:
If the same pool is being run across multiple lanes, it should be listed as follows:
- Row 1: Lane 1,2,3,4,5,6,7,8, Sample 1
- Row 2: Lane 1,2,3,4,5,6,7,8, Sample 2
📌 Note: The number of lanes depends on your sequencing setup. Be sure to confirm the correct lane allocation for your specific sequencing run. Information about different flow cells can be found here: Illumina specifications for the NovaSeq X and NextSeq 2000
4. Loading Concentration & Sample Sheet
You must prepare your pool at the final concentration recommended by Illumina for your specific library type. This is entirely the customer’s responsibility, as we do not have specific information about your library.
5. Submission Requirements
- Final Concentration: Your pool should be diluted to 2nM using RSB buffer (Illumina). Important: the RSB buffer is not included in the NovaSeq X Plus sequencing kit. For detailed instructions, refer to "Diluting Libraries Illumina Novaseq X Plus" or NextSeq 2000
- Sample Sheet: Include a corresponding sample sheet and verify that the indexes do not clash.
🔹 Customer Responsibility: The customer is responsible for ensuring the accuracy of the sample sheet information and pooling. We will not be able to troubleshoot your sample sheet if any issues occur.
6. Sequencing Process Timeline
- Open a project and coordinate with the responsible person.always give us a date that you will come with your pool.
- Approve the quote, after which your sequencing kit will be ordered.
- Prepare your pool and deliver it to us.
📌 Estimated Turnaround Time: Once we receive your pool, it typically takes one month to deliver your sequencing data.
Information about different flow cells can be found here: Illumina specifications for the NovaSeq X/X Plus | Illumina Knowledge
