Sequencing Only
CTG provides service for sequencing of user-prepared libraries on NovaSeq 6000 and NextSeq2000. Sequencing on NovaSeq X Plus will be available shortly.
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 requested Illumina platform.
- NovaSeq libraries need to have unique dual indexes.
Read about unique dual indexes here (opens in new tab).
- 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 EB buffer (10mM Tris-HCl, pH 8.5).
- 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 6000
| Pool volume | SP/S1 – 220 µl (minimum 110 µl) S2 – 320 µl (minimum 160 µl) S4 – 640 µl (minimum 320 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 some guidance but cannot take responsibility for this optimization. |
| Indexes |
Unique dual indexing is required for NovaSeq (unless the manufacturer of your library preparation kit specifies differently) |
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. |
NovaSeq X Plus
| Pool volume | 50 µl (minimum 30 µl) per lane, flowcells come with 8 lanes. |
| 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. |
Data Analysis and Delivery
After sequencing is completed, you will receive a delivery mail containing instructions on how to access your data through our delivery server.
Please note that the Sequencing Only analysis pipeline does not include any downstream data processing and/or interpretation other than generation of FASTQ-files and basic QC metrics.
The CTG Sequencing Only data delivery will include:
| Quality Control files |
FastQC, MultiQC. |
| FASTQ files |
Demultiplexed data files generated from the Illmina sequencing run. Requires SampleSheet with index sequence information. If proper index sequence information is not supplied we will generate undetermined FASTQ files. |
After download, we recommend checking the data integrity using the md5sum-files provided.
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!
Contact CTG Bioinformatics
If you have any questions with regards to the bioinformatic processing of your data please contact CTG Bioinformatics Team.
Contact for service
Phone: +46730753563
E-mail: CTGservice [at] med [dot] lu [dot] se (CTGservice[at]med[dot]lu[dot]se)
