Raising the bar in scalability and accuracy
Watchmaker DNA Library Prep Kits with Fragmentation enable highly sensitive clinical and translational applications to access meaningful insights from a broad range of biological sample types, including ultra-low inputs and FFPE, with an unparalleled combination of accuracy and scalability.
The workflow harnesses the process benefits of enzymatic fragmentation, such as ease of automation, increased scalability, and preservation of low input samples, while mitigating the formation of associated library preparation artifacts, including false chimeric reads and hairpin artifacts, that can convolute variant calling. Further, use of the Equinox polymerase delivers ultra-high-fidelity, low-bias library amplification.
Key Features and Benefits
- Up to a 90% reduction in sequence artifacts improves assay accuracy – critical for highly sensitive applications
- Improved library amplification polymerase error rates and even UMI family coverage enable rare mutation detection
- Robust fragmentation and library prep efficiency support the use of clinically relevant sample types, such as low input and degraded
- Highly tunable fragmentation delivers consistent library sizes over a wide input range (< 1 ng to 500 ng) and between library batches
- Streamlined workflow delivers PCR-free libraries in under 90 minutes and scales easily to high sample numbers and automation platforms
- Uniform sequence coverage improves sequencing efficiency
- Somatic mutation calling and other low-frequency variant detection NGS assays, including those utilizing challenging samples such as FFPE
- Inherited disease sequencing
- Human whole genome sequencing (WGS), including PCR-free
- Whole exome sequencing (WES)
- Single cell analysis
- Metagenomic analysis
- Bulk RNA sequencing (using cDNA as input)
- Viral genome sequencing
- Microbial WGS
Figure 1. Simplified and easily automated workflow. The Watchmaker DNA Library Prep Kit with Fragmentation simplifies library construction by using combined enzymatic steps that were designed with automation in mind. Generous overages ensure sufficient fill volumes for automated library preparation, and the use of a ligation master mix reduces hands on time.
Key Performance Data
Reduced sequence artifacts enables high-sensitivity applications
The Watchmaker DNA Library Prep Kit with Fragmentation alleviates many issues associated with sonication, delivering ease of automation and improved scalability, while also mitigating the formation of sequence artifacts, such as false chimeric reads and false SNVs resulting from hairpin artifacts1, that are often associated with enzymatic fragmentation. These artifacts convolute the identification of true structural and single nucleotide variants in a sample and especially impact highly sensitive applications, such as low-allele-fraction variant calling.
1Thomas Gregory, Apollinaire Ngankeu, Shelley Orwick, Esko A Kautto, Jennifer A Woyach, John C Byrd, James S Blachly, Characterization and mitigation of fragmentation enzyme-induced dual stranded artifacts, NAR Genomics and Bioinformatics, Volume 2, Issue 4, December 2020, lqaa070, https://doi.org/10.1093/nargab/lqaa070
Ultra-high fidelity amplification improves accuracy
The Equinox Library Amplification Master Mix contains a proprietary proofreading polymerase optimized for ultra-high fidelity amplification delivering a 40% reduction in overall polymerase error rate in comparison to KAPA HiFi HotStart. This enables sensitive variant detection by minimizing overall error rates and reducing false variant calls. The significant reduction in C>T substitutions is particularly important as this mutation is associated with the spontaneous deamination of methylated cytosine to uracil, and is also one of the most common mutation types in cancers.
Robust performance improves utility of clinically relevant sample types
Challenging clinically relevant sample types have historically been difficult to reproducibly process. Vanishingly small input amounts raise the issue of inherent sample loss when using sonication to shear genomic DNA. Formalin-fixed paraffin-embedded (FFPE) samples, while critical to oncology research, are typically highly damaged as a result of the fixation process. The Watchmaker DNA Library Prep Kit with Fragmentation delivers high-quality libraries, exhibiting exceedingly small amounts of adapter-dimer contamination, with ultra-low input amounts and poor sample qualities, improving researchers’ ability to derive meaningful biological interpretations from sequence data.
Tunable fragmentation ensures compatibility with a broad range of applications
The Watchmaker DNA Library Prep Kit with Fragmentation delivers highly tunable DNA fragmentation through modulation of both reaction time and temperature. The wide range of attainable library distributions makes the workflow broadly compatible with a variety of applications and sequencing read lengths.
Uniform sequence coverage improves sequencing economy
Watchmaker DNA Library Prep Kits with Fragmentation delivers even sequence coverage uniformity across complex genomes. This can reduce the amount of overall sequencing – and associated costs – needed to achieve desired coverage depths for all regions of interest.