Illumina DNA PCR-Free

Method for shotgun DNA libraries used for whole genome sequencing and metagenomics.

Illumina’s new tagmentation-based DNA PCR-free library preparation is the method of choice for generating high quality DNA sequencing libraries. PCR-free libraries have better coverage of GC-rich regions compared to PCR-based methods and the reads are more evenly distributed over the genome.

This method is recommended when:

  • Insert sizes approx. 350 bp are suitable for your application.
  • You do not have enough DNA for the TruSeq PCR-free protocol

Sample requirements

  • Sample type: DNA
  • Volume: >30 µL (max 100 µL per well). 2 µL of the sample will be used for our initial quality checks, so please account for this when sending us samples.
  • Amount of material needed for 1 library preparation
    • Standard input protocol: 300 ng (works down to >200 ng). Recommended for larger genomes or metagenomes. Conc >15ng/µL.
    • Low input protocol: 25-200 ng (>100 ng recommended); conc >5 ng/µL (NB: We are currently optimising automation for this protocol, so it is not being offered at present).
  • Make sure that the DNA sample does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the tagmentation reaction and result in unexpected library insert sizes.

How to evaluate the sample quality

We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples.

Checking the concentration
Please use fluorometric measurements (e.g. Qubit, Quant-it), not absorbance measurements (Nanodrop, spectrophotometer).

If your samples are below the required thresholds, please get in touch.

What we do with your samples

Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the samples meet our requirements.

If the samples fail this quality control step, we will contact you to discuss possible options. Should you choose to proceed with samples not fulfilling the criteria it will be at your own risk, NGI will only make one attempt at library preparation and if the prep does not work you will have to pay for the library preparation anyway.

If the samples pass reception control, we will inform you and the samples will be queued for library preparation.

Library preparation

Library QC and sequencing

We evaluate the concentration of the library and inform you of the QC status of each sample. Once the libraries have passed this QC step, they are normalised, pooled, and queued for sequencing.

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

Expected results

PCR-free libraries are well suited for deep sequencing to achieve high coverage genomes. The library preparation method shows a significantly better coverage of GC-rich regions compared to PCR-based methods and the reads are more evenly distributed over the genome.

Last Updated: 29th February 2024

Please read our sample submission instructions before sending samples:

Sample Submission Guidelines
Relevant Technologies
Bioinformatics Pipelines
Method Status


We are routinely running this method. Please visit the Order Portal to place an order.