Illumina 16S sequencing

Sequencing of the 16S gene to study bacterial diversity and composition in a sample.

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 region of interest (ROI). The locus-specific primers have 5′- overhang handles which are used in a second PCR to introduce sample-specific index sequences. This ensures that individual samples can be identified after sequencing. A popular application is sequencing hyper-variable parts of bacterial genes (e.g. 16S), which enables deconvolution of the bacterial species composition of many environmental samples in parallel.

This application is for 16S sequencing, where a specific primer pair is used to amplify a hyper-variable part (V3/V4) of the bacterial 16S gene. For users interested in 16S data, we can do the whole process (both PCR-steps) for you. If you have already performed a PCR of your specific ROI, we also offer Illumina custom amplicon sequencing.

You would want to use this protocol when

  • You want to get information on the presence of bacterial populations in a sample (based on 16S amplification performed at NGI)
  • You are not interested in archaea (you would need to choose the other amplicon option for that)
  • We only have 16S V3/V4 primers using 341F/805R

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: gDNA
  • Sample Amount: normalized, at a concentration of 0.25 ng/μl of bacterial gDNA (your input might differ if you have high amounts of contaminating DNA of non-bacterial origin)
  • Sample Volume: 12 μl
  • Sample QC: concentration measurements using Qubit HS DNA
  • Maximum number of samples/plate: 94, leave the last 2 wells in the last column (G12-H12) EMPTY (please do not add water or buffer)

Before starting the project we would like you to perform a test PCR on a few of your samples.

We will send you an aliquot of our PCR1 primers and want you to test them using a couple of representative samples according to the protocol below.

PCR reaction
– 4 μL sample (total 1ng)
– 10μL KAPA 2x
– 0.5μL BSA (20mg/uL)
– 4.5μL water
– 2μL Fwd primer and Rev primer (mixed)

PCR setup
1. Denaturation @ 98C°, 2min
2. Denaturation @ 98 C°, 20 s
3. Annealing @ 54˚C, 20 s
4. Elongation @ 72C°, 15 s
5. GO TO step 2, 28 cycles
6. Final elongation @ 72C°, 2 min

Run products on a 1% agarose gel to make sure there is only clear one band at ca 500bp. Please submit this gel photo to us together with the rest of the sample information.

How to evaluate the sample quality

We recommend evaluation of sample concentrations using Qubit and checking the size of DNA after test amplification on agarose gel electrophoresis or TapeStation/Fragment Analyzer.

You will receive primers and detailed instruction for the test PCR together with the plates for sample submission.

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 receiving your samples we will perform a first, target-specific PCR on 4 μl of your sample. We do not measure the DNA concentration in the samples, so be sure you are within the limits for acceptable sample concentration. After the first PCR, we purify the product using MagSI beads. During this purification we remove left-over adapters and concentrate the sample. 

A second PCR step adds indexes and Illumina flowcell adapters to the samples, followed by another round of bead-based library purification.

We will run a positive and negative control on every plate, to ensure that we fullfill our standards for library prep.

Library QC and sequencing

At the end of the 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 some 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 that show the composition of microbial populations. However, the library preparation 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 microbial DNA content due to non-microbial DNA 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.