Illumina TruSeq DNA PCR-free
Gold standard method for shotgun DNA libraries used for whole genome sequencing and metagenomics.
Illumina TruSeq PCR-free library preparation is the method of choice for generating the highest quality DNA sequencing libraries. TruSeq 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:
- You have ample starting material (>1000 ng).
- You want to adjust the insert size of the library. Multiple insert sizes are currently available: 350 bp (standard), and 550 bp.
Sample requirements
- Sample type: DNA
- Concentration: 50-300 ng/µL
- Volume requirement NGI Stockholm: >15 µL (max 100 µL per well). Approx. 2 µL of the sample will be used for our initial quality checks, so please account for this when sending us samples.
- Volume requirement NGI Uppsala: 25-50 µl.
Please contact a coordinator if the sample requirements cannot be fulfilled. - Amount of material used for prep: 1 µg (350 bp) or 2 µg (550 bp)
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).
Checking the quality and purity
The DNA should be in water or EB buffer, of high quality, and ideally have an OD260/280 of 1.8-2.
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
DNA is sheared on a Covaris sonicator followed by a size selection using AMPure beads. Illumina adapters with sample-specific barcode sequences are ligated.
Library QC and sequencing
We evaluate the concentration of the library using qPCR 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
TruSeq 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: 3rd April 2024