No duplicates
Without an amplification step, no PCR duplicates are generated. The entire sequencing output represents unique molecules: no duplicates and more usable reads at the same capacity.
Twist Bioscience · NGS portfolio
PCR-Free WGS Library Prep Kit for faithful native-genome representation
Library preparation for whole-genome sequencing without a PCR amplification step. No PCR duplicates, significantly reduced GC bias and longer inserts for better detection of structural variants.
- PCR-free No duplicates, no PCR errors
- 40–300 ng Wide DNA input range
- ~400 bp Consistent median insert size
What is PCR-free WGS
Library preparation without PCR amplification
Standard WGS workflows include a PCR amplification step. It raises the yield from low-input samples, but it also introduces duplicates and GC bias and distorts coverage in repetitive and GC-rich regions. The Twist PCR-Free WGS Library Prep Kit skips this step entirely: the DNA undergoes enzymatic fragmentation and adapter ligation, and that is it. No PCR.
The result is a library that faithfully reflects the distribution of the original genome: better coverage of GC- and AT-rich regions, longer inserts (median ~400 bp) and more sensitive detection of structural variants and repetitive regions. The kit works with 40–300 ng of input DNA.
Key advantages
Why a PCR-free workflow
Minimal GC bias
Normalised coverage stays close to 1.0 across the 10–90% GC content range, with no excessive over- or under-sequencing of GC-rich regions.
Reduced dropout in AT-rich regions
The PCR-free workflow preserves the native representation of the genome, eliminates dropout in AT-rich regions and improves coverage uniformity across difficult regions of the genome.
Longer inserts for structural variants
A median insert size of ~400 bp improves mapping accuracy and the sensitivity of structural variant detection. Less bias towards short fragments.
Consistent yield at low input
Optimised ligation chemistry maintains library yield even at the lowest tested input of 37.5 ng DNA, with the library above 2 nM.
How it works
Workflow in three steps
01
Enzymatic fragmentation
Native DNA is fragmented at 25 °C for 15 min. No sonication, no covalent modifications: just clean enzymatic chemistry with consistent results across inputs.
02
Adapter ligation
Optimised Twist ligase ensures high adapter ligation efficiency while limiting the introduction of bias. All without subsequent PCR amplification.
03
QC and sequencing on NovaSeq X
qPCR quantification of the library (e.g. Kapa Library Quantification), pooling of up to 1,536 samples and sequencing on NovaSeq X. Loading threshold ≥ 2 nM, met even at the lowest tested input of 37.5 ng.
Measured data
Consistent results across inputs
Library concentration, insert size and coverage uniformity across GC content remain stable over the entire tested range from 37.5 ng to 300 ng of DNA.
Library concentration vs. DNA input
~15 nM at 300 ng input, ~4–5 nM at 75 ng and ~2.3 nM at 37.5 ng. Always above the 2 nM threshold.
Library quality
Consistent library size of ~3.3 × 10¹⁰ molecules at 300 ng and ~2.2 × 10¹⁰ at 37.5 ng. High conversion yield.
Normalised coverage vs. GC content
Normalised coverage close to 1.0 across the 10–90% GC range for all three inputs. No bias at the extremes of GC content.
Watch
PCR-Free WGS Kit in action
A short video introduces the workflow of the Twist PCR-Free WGS Library Prep Kit, from fragmentation to loading on NovaSeq X.
Let's start
Interested in the Twist PCR-Free WGS Kit?
Talk to us about the right use for your application. We will help you test and validate the protocol directly in your laboratory, including technical support.