Aviti sequencing
NGI can sequence user-prepared libraries on the Aviti platform. User-prepared libraries orders can only be sequenced on whole flowcells.
Sample requirements
We use the Cloudbreak FS flowcells, meaning that most Illumina compatible libraries can be sequenced directly without the need for any conversion.
We only accept pools of your libraries, meaning you need to normalise the libraries and pool them yourself to whatever ratio you need in order to give the amount of sequencing reads required per sample.
The 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.
Please get in touch if the concentration of your pool is much lower than 10 nM. We also 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.
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 gives you a very exact measure of the concentration but also provides 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 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.
For the Aviti, indexes are reported in forward (i7) – forward (i5) orientation which differs from Illumina where i5 is reported in the reverse complement orientation.
Aviti runs use indexed PhiX libraries, so the following combinations should be avoided in any pools sequenced on the Aviti flowcells.
- TGTGTCGACA-TGTCTGACAG
- GCACATAGTC-GACTACTAGC
- ATGTCGCTAG-CTAGCTCGTA
- CACAGATCGT-ACGAGAGTCT
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 (if required).
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: 27th May 2026