Library Preparation Enzymatic Fragmentation Kit 2.0

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.

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.

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.

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.

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.