Illumina amplicon sequencing

Sequencing of PCR amplicons to study genetic variation within small target regions.

Amplicon sequencing involves sequencing of PCR-amplified loci across large sets of samples. The method involves two PCR-steps, the first using locus-specific primers to amplify a specific locus. The locus-specific primers have 5′- overhang handles which are used in a second PCR to introduce sample specific index sequences allowing individual samples to be identified after sequencing.

This application is for custom amplicons where one or more user defined loci are amplified prior to submission. We also offer Illumina 16S sequencing where there is no need for users to perform a first amplification.

You would want to use this protocol when:

This method requires you to do the 1st PCR. You need to contact us before ordering the PCR primers. This method is primarily of interest when:

  • Your region of interest is specific to your project, i.e. we don’t perform the first PCR.
  • You have strongly variable input concentrations
  • You need to use a different PCR setup (reaction sizes, polymerases, etc) than supported by our protocol
  • Your region of interest is difficult to amplify and/or the PCR requires gel purification of specific bands.

Sample requirements

We do not perform any kind of reception control, all samples delivered will be used for library prep as is. Therefore, the responsibility is yours to provide good sample quality and the correct quantity.

  • Sample Type: PCR fragment of interest using compatible primers
  • Sample Amount: 0.25-0.8 ng/μl
  • Sample Volume: 10 μl
  • Sample QC: concentration measurements using Qubit HS DNA, preferred: gel of PCR product for some samples
  • Maximum number of samples/plate: 95

How to evaluate the sample quality

We recommend checking sample concentrations using the Qubit HS DNA kit and evaluation of the amplicon fragment sizes via agarose gel electrophoresis or TapeStation/Fragment Analyzer.

If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.

What we do with your samples

Library preparation

When we receive your samples we will perform only the second PCR, adding the indexes. The first PCR, performed by you, therefore needs to have been done with compatible primers (please contact us in advance). We do not measure the DNA concentration of material, so be sure you are within the limits for acceptable sample concentration. This is followed by a bead-based library purification using MagSI beads.

We will run a negative control on each plate, to exclude contaminations during processing at NGI.

Library QC and sequencing

At the end of library preparation, we evaluate the yield obtained and we will also determine the size distribution of a subset of libraries generated. We will inform you of the QC status of the library preparation and whether any samples did not meet required concentration levels. Once the libraries have passed this QC step, they are queued for sequencing.

The sequencing will be carried out following the setup stated in the agreement.

Expected results

We have successfully generated and sequenced libraries in a number of projects, using this protocol. The library preparation, however, strongly depends on the correct concentration of the input material. Furthermore, different sample types might have issues due to PCR inhibitors in the samples or incorrect measurements of DNA content due to contaminations. We cannot check these issues in your samples. If a library preparation fails but our positive control worked we will not repeat the preparation.


Relevant Technologies
Bioinformatics Pipelines
Method Status


We are currently testing this method. Please contact us to find out more.