Enhanced Enzymatic Fragmentation
The Twist Library Preparation Enzymatic Fragmentation (EF) Kit 2.0 is built to help you achieve a more efficient sequencing pipeline and obtain more accurate Next-Generation Sequencing (NGS) results. Compared to the original Twist Library Preparation EF Kit 1.0, this updated kit has several benefits. In addition to new fragmentation and ligation modules, the upgraded kit now includes the Equinox Library Prep Amp Mix. This hot-start enzyme formulation has a lower error rate* and high efficiency at low input volumes—making it ideal for samples such as FFPE, where high efficiency and yield are necessary.
The kit’s streamlined workflow combines library preparation steps into a single-tube reaction which, alongside a low chimera rate and Twist’s highly uniform target enrichment workflow, translates into a better quality library preparation and sequencing result.
*Relative to Twist Enzymatic Fragmentation 1.0
Library Preparation Workflow
The Twist Library Preparation EF Kit 2.0 includes the reagents required for end-repair, dA-tailing, adapter ligation, and library amplification. This Kit also incorporates the enzymes for fragmentation of gDNA samples and allows for tunable insert sizes. Following core library construction, either full-length or universal adapters can be used to suit your application needs.
KEY BENEFITS
- Flexible DNA sample input from 1 ng – 500 ng
- Convert DNA samples into robust, amplified libraries in three hours
- Single-tube library prep for improved efficiency and consistent results
- Hot-start enzyme formulation with high efficiency at low input volumes
- High range of GC content tolerance (20 – 80%) during library preparation
- Improved error rate compared to a frequently used polymerase
- Pairs with Twist’s Target Enrichment workflow to enable highly uniform amplification
- Higher confidence in true-chimera detection
- Consistent yield from high-complexity libraries delivers on sensitivity and specificity
Stay On-Target
The on-target rate for the Twist Library Preparation EF Kit 2.0 is equivalent to the on-target rate achieved with the Twist Library Preparation EF Kit 1.0. Shown here is a comparison of the fraction of on-target calls under different hybridization conditions.
Target enrichment was performed with the Twist Core Exome Panel and Twist’s Universal Adapter system. Sequencing was performed on NextSeq® 550 or 2000 platforms. Data were down-sampled to 150x of target size and analyzed using Picard Metrics. Error bars represent the standard deviation between replicates.
Reduce Chimera Rates to Reduce Sequencing Error
During Library Preparation, minimizing inappropriate DNA ligation or recombination minimizes chimera-derived sequencing errors. The upgraded Twist Library Preparation EF Kit 2.0 provides a significantly decreased fraction of chimera reads, shown here against the Twist Library Preparation EF Kit 1.0. It also shows equivalent chimera rates to library preparation with mechanical fragmentation.
NGS libraries were generated with the Twist Library Preparation Kit with Enzymatic Fragmentation 1.0, Twist Enzymatic Kit with Enzymatic Fragmentation 2.0, or Twist Library Preparation with Mechanical Fragmentation kit. Target enrichment was performed with Twist Core Exome Panel and Twist’s Universal Adapter system. Sequencing was performed on a NextSeq® 2000 platform. Data were down-sampled to 150x of target size and the Picard metric PCT_CHIMERAS is reported. Error bars represent the standard deviation between replicates.
Consistent Performance From Run to Run
Repeatable performance ensures confidence in results. When experimental conditions are held constant, the Twist Library Preparation EF Kit 2.0 produces a robust, consistent DNA library fragment size. Shown here are 8 different electropherograms of NGS libraries generated with the kit. The overlap of the fluorescent curves demonstrates the consistency in library preparation that can be achieved from run to run.
NGS libraries were generated with the Twist Library Preparation EF Kit 2.0 and Twist’s Universal Adapter system. 50 ng of high-quality gDNA was fragmented for 20 minutes at 37°C. 6 cycles of PCR were utilized for amplification. Samples were analyzed with an Agilent DNA 7500 assay and results overlaid in Expert 2100 software.
Tune fragment size to fit your experimental needs
With the Twist Library Preparation EF Kit 2.0, DNA library fragment size can be tuned to your specific needs by simply adjusting fragmentation time. Shown here are four electropherograms of NGS libraries generated using differing fragmentation times.
NGS libraries were generated with the Twist Library Preparation EF Kit 2.0 and Twist’s Universal Adapter system. 50 ng of high-quality gDNA was fragmented for various times at 37°C. 6 cycles of PCR were utilized for amplification. Samples were analyzed with an Agilent DNA 7500 assay and results overlaid in Expert 2100 software.
Improved library amplification to minimize sequencing error
Errors during amplification are unavoidable, but they need not be common. The upgraded Twist Library Preparation EF Kit 2.0 includes the Equinox Master Mix, featuring a high-fidelity hot-start enzyme. Compared to a previously recommended and commonly used polymerase, the Equinox Master Mix demonstrates a lower error rate with fewer misincorporated bases. This means more accurate amplification and ultimately sequencing data you can trust.
Base misincorporation events across numerous pairs of mismatched bases were measured with a proprietary NGS-based assay. Values presented represent an average error rate of all misincorporation events.
PRODUCT SKUS – Twist Library Preparation EF Kit 2.0
104206: Twist Library Preparation EF Kit 2.0, 16 Samples
104207: Twist Library Preparation EF Kit 2.0, 96 Samples
Reagents, enzymes, and purification beads required for enzymatic fragmentation of gDNA, library construction and amplification
