Illumina sequencing
NGI can sequence user-prepared libraries on all Illumina instruments. User-prepared libraries orders can only be whole-lane orders. Libraries with custom setups and or custom primers can only be whole flow cells.
Sample requirements
We only accept pools of your libraries, meaning you need to normalise and pool them yourself to whatever ratio you need in order to give the amount of sequencing reads required per sample.
In case you are making pools with very few libraries it’s extra important that you read and understand the pooling guidelines, especially for sequencing on Illuminas new 2-color systems such as NovaSeq and NextSeq. You can read more about this in the last section (Other considerations when choosing indexes) on this page.
Minimum concentration for a library pool varies depending on the type of flowcell you have ordered, we generally recommend you deliver a pool with a concentration of ca 10 nM. The required volume also varies depending on flowcell, see summary in the list below:
- MiSeq/NextSeq: at least 20 μL of 10 nM library pool
- NovaSeq X and 6000 (clustering on separate lanes): at least 20 μL of 10 nM library pool
- NovaSeq 6000 (clustering on whole flowcell): at least 70 μL of 10 nM library pool
Please get in touch if the concentration of your pool is much lower than 10 nM. We also like to ask you to dilute the pool if concentration is too high, around 10-20 nM is a good range.
Approx 2 μL of the sample will be used for our initial quality checks, so please account for this when sending us samples.
The range of fragment sizes within the library pool should ideally be larger than the sequencing read length and less than ca 800 bp, no traces of primer dimers etc should remain. Feel free to contact us if you are unsure about how to evaluate the electropherogram.
Custom primers
It is possible to use custom sequencing primers or a combination of custom primers and Illumina primers provided in the reagent cartridge. Please supply a minimum of 30 μL of each custom primer at a stock concentration of 100 μM. It must be clearly specified in your order if you require custom primers, how many, and for which read or reads (e.g. R1, i7, i5, R2). In some cases this means an additional kit being used which will require an extra fee for sequencing.
When using custom primers you need to order a full flowcell.
Please contact us if you have questions regarding custom primers.
How to evaluate the sample quality
You need to determine the molar concentration of each single library in the pool and then pool them to whatever molar ratio you require. In addition to this you also need to submit the following information to us before delivering your sample/samples:
- Concentration of the pool, either using traditional spectrophotometric or fluorometric methods, but preferably using qPCR (e.g. using the KAPA Library Quantification kit for Illumina). Doing qPCR not only give you a very exact measure of the concentration but also provide a confirmation that the libraries contain the correct adapters and that the libraries can actually cluster on the flowcell. If for example a concentration measurement on Qubit is much higher than the qPCR concentration it may be an indication that something has gone wrong in library preparation. qPCR will directly give you the molar concentration while a spectrophotometer or fluorometer will give you a concentration in ng/μL that needs to be converted to nM by using the average fragment length you get from the electropherogram (see next point).
- We require Bioanalyzer (or similar) traces of the submitted library pool (it’s important you submit the electropherogram of the completed pool and NOT the individual libraries). The scale on the x-axis must be in basepairs. This is used to calculate the molar concentration in case you did not use qPCR, but it will also show important information about the fragment size distribution of all the libraries in the pool. An important thing to look for is adapter dimer content, in case there is a large amount of these short adapter containing fragments there is a risk they will totally take over during clustering due to their small size. You can find more information here.
- In addition to the information above you also need to submit information about the indexes used for the libraries in your pool so we can perform demultiplexing of the data. All this information should be added in the Excel template sent to you once we have accepted your order.
If you are not able to provide a Bioanalyzer trace, or your samples are below the required thresholds, please get in touch.
What we do with your samples
When receiving your library pool, we first determine the concentration by qPCR measurement plus reviewing the fragment size information submitted by you. If anything does not fulfil our criteria you will be contacted and we can discuss options, or let you decide to proceed with sequencing anyway. Once the project has a go ahead for sequencing we denature and dilute the pool and add the PhiX spike-in. We normally add 1% PhiX to all Illumina runs, but for libraries with low base-complexity (such as amplicons) a higher PhiX spike-in is recommended (usually ca 10%).
Expected results
Please see respective sequencing platform under “relevant technologies” in the sidebar to the right.
Bioinformatics
We will follow our bioinformatics analysis guidelines to do the sequencing run QC and the demultiplexing.
Sequencing quality control is done based on yield and quality scores (exact criteria varies depending on sequencing platform and flowcell), and in case any of these criteria is not fulfilled we will re-sequence any pool that has passed the sample quality control.
Last Updated: 12th March 2024