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Sequencing Only

CTG provides service for sequencing of user-prepared libraries on NovaSeq 6000, NextSeq2000 and MiSeq. The SeqOnly analysis pipeline does not include any downstream data processing and/or interpretation other than basic QC metrics and (optional) generation of FASTQ-files.


General information before starting a Sequencing Only project
 

Please read through these following steps

  • Your libraries need to be compatible with requested Illumina platform
     
  • NovaSeq libraries needs 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 machine and sequencing kit specific
     
  • Illumina has guidelines about library loading concentration but they 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 for Illumina if you want demultiplexing (FASTQ files)
    - SampleSheets is made with Illumina Experiment Manager (IEM) software (opens in new tab)
    - a template can be provided by CTG if you have problems with IEM

Library concentration and quality check


We recommend you to do the following steps before and after pooling your libraries:

  1. Verify concentration of libraries using a fluorometric assay
  2. Perform quality and size calculation of libraries on a micro-capillary gel electrophoresis system.
    Make sure no traces of primer dimers etc remains
  3. Calculate molarity of libraries based on the concentration and library size
  4. Dilute and pool your libraries in EB buffer (10mM Tris-HCl, pH 8.5)
  5. Verify concentration of pool using a fluorometric assay before delivery
  6. Calculate molarity of diluted pool using pool concentration and average library size

Sample Requirements

Sample requirements - NovaSeq

Pool volume SP/S1 – 220 ul (minimum 110 ul)
S2 – 320 ul (minimum 160 ul)
S4 – 640 ul (minimum 320 ul)
Pool molarity Library specific

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 For NovaSeq unique dual index is needed (if not producer of your library preparation kit is stating differently for NovaSeq)

If demultiplexing (FASTQ files) is needed notice that reverse compliment on i5 indices is needed in the SampleSheet for NovaSeq kits v1.5. The Illumina IEM software will provide you with the correct index sequences for Illumina kits.

Sample requirements - NextSeq 2000

Pool volume 60 ul (minimum 30 ul)
Pool molarity

Library specific

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.
The pool need to be diluted to loading concentration with RSB/Tween buffer, CTG can provide this buffer. Please contact CTG for more detailed information.
 

CTG may give you guidance but cannot take responsibility for this optimization.

Indexes If demultiplexing (FASTQ files) is needed you must supply a Illumina compatible SampleSheet. The Illumina IEM software (or the Illiumina BaseSpace Hub) will provide you with the correct index sequences for Illumina kits.

Sample requirements - MiSeq

Pool volume 50 ul
Pool molarity 4 nM
Indexes Make sure your indices and adapters are Illumina compatible. The Illumina IEM software will provide you with the correct index sequences for Illumina kits for Miseq.

Data Analysis and Delivery


You will receive a delivery mail containing:

  • a report including run conditions
  • instructions on how to access your data through our delivery server


After download, we recommend you to check data integrity using the md5sum-files provided.

After sequencing delivery, data will be kept by CTG for 1 month and subsequently deleted. We strongly recommend you to backup your data, i.e. that have at least two copies.  CTG can not help you if your data gets corrupt or lost!

Please note that the SeqOnly analysis pipeline does not include any downstream data processing and/or interpretation other than basic QC metrics and (optional) generation of FASTQ-files.
 

The CTG SeqOnly data delivery will include

Raw Data Complete Illumina Sequencing RunFolder:
sequencing basecall (BCL), run information and InterOp statistics files. 
Quality Control (QC) files FastQC, MuliQC and sequencing run InterOp statistics
FASTQ files
(optional)
Demultiplexed data files generated from the Illumina sequencing run.

Requires SampleSheet with proper index and adapter sequence information. If index sequence information is not properly supplied you can perform your own demultiplexing using the Raw Data.

Contact for service

CTGservice [at] med [dot] lu [dot] setarget="_self"

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