WATCHMAKER DNA LIBRARY PREP KITS WITH FRAGMENTATION

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Accuracy at Scale

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.

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 Key Features and Benefits

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Watchmaker DNA Library Prep Kits

High-efficiency library prep
from cfDNA and more.

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Applications

  • 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
  • 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
  • Metagenomic analysis
  • Bulk RNA sequencing (using cDNA as input)
  • Viral genome sequencing
  • Microbial WGS

Workflow

Simplified and easily automated workflow.
Figure 1. Simplified and easily automated workflow. The Watchmaker DNA Library Prep Kit with Fragmentation simplifies library construction by using combined enzymatic steps (FRAG/AT) and a ready-to-use ligation master mix to reduce hands-on time compared to a widely used enzymatic fragmentation kit (Supplier A). The Watchmaker kit was designed with automation in mind with generous overages to ensure sufficient fill volumes for automated library preparation.

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 artifacts, 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 calling1.

1 Thomas 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

Figure 2A. Reduced chimeric reads enable identification of true structural variants. Libraries were prepared in duplicate from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared.
Figure 2B. Reduced hairpin artifacts improve assay sensitivity. Reduced hairpin artifacts improve assay sensitivity. Libraries were prepared in duplicate from 10, 50, and 100 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared using a ligation-based library preparation workflow and 1 and 200 ng of human genomic DNA input. Data bars represent the mean of duplicate libraries across all DNA input amounts assessed per library preparation workflow, and error bars show the standard deviation.
Reduced sequence artifacts enables high-sensitivity applications
Figure 2A. Reduced chimeric reads enable identification of true structural variants. Libraries were prepared in duplicate from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared.
Figure 2A. Reduced chimeric reads enable identification of true structural variants. Libraries were prepared in duplicate from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared.
Figure 2B. Reduced hairpin artifacts improve assay sensitivity. Reduced hairpin artifacts improve assay sensitivity. Libraries were prepared in duplicate from 10, 50, and 100 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared using a ligation-based library preparation workflow and 1 and 200 ng of human genomic DNA input. Data bars represent the mean of duplicate libraries across all DNA input amounts assessed per library preparation workflow, and error bars show the standard deviation.
Figure 2B. Reduced hairpin artifacts improve assay sensitivity. Reduced hairpin artifacts improve assay sensitivity. Libraries were prepared in duplicate from 10, 50, and 100 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation, or two widely used enzymatic fragmentation kits per manufacturer’s recommendations. A sonication control library set was also prepared using a ligation-based library preparation workflow and 1 and 200 ng of human genomic DNA input. Data bars represent the mean of duplicate libraries across all DNA input amounts assessed per library preparation workflow, and error bars show the standard deviation.
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 a high-fidelity PCR HotStart DNA Polymerase. 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.

Figure 3. Up to 40% reduction in overall polymerase error rate. Error rates of the Equinox Library Amplification Master Mix and a widely used high-fidelity amplification kit were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. Base substitution profiles were examined in triplicate over 5.4 million G/C incorporation events and 4.0 million A/T incorporation events.
Ultra-high fidelity amplification improves accuracy
Figure 3. Up to 40% reduction in overall polymerase error rate. Error rates of the Equinox Library Amplification Master Mix and a widely used high-fidelity amplification kit were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. Base substitution profiles were examined in triplicate over 5.4 million G/C incorporation events and 4.0 million A/T incorporation events.
Figure 3. Up to 40% reduction in overall polymerase error rate. Error rates of the Equinox Library Amplification Master Mix and a widely used high-fidelity amplification kit were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. Base substitution profiles were examined in triplicate over 5.4 million G/C incorporation events and 4.0 million A/T incorporation events.
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.

Figure 4A. Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
Figure 4B. Efficient library preparation across a wide range of sample qualities. Libraries were constructed in duplicate from 25 ng of either high-quality or formalin-compromised DNA of moderate to severe damage (HD799 and HD803, respectively; Horizon Discovery) using the Watchmaker DNA Library Prep Kit or two competitor enzymatic fragmentation kits. High-quality DNA fragmentation times were selected to target final library sizes of approximately 500 bp per manufacturer’s published recommendations (5, 10, and 10 minutes at 37℃, respectively). Damaged DNA fragmentation times were fixed at 10 minutes at 37℃. All libraries were amplified for 6 PCR cycles. Final library distributions and yields were assessed using a D1000 assay by TapeStation (Agilent).
Robust performance improves utility of clinically relevant sample types
Figure 4A. Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
Figure 4A. Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
Figure 4B. Efficient library preparation across a wide range of sample qualities. Libraries were constructed in duplicate from 25 ng of either high-quality or formalin-compromised DNA of moderate to severe damage (HD799 and HD803, respectively; Horizon Discovery) using the Watchmaker DNA Library Prep Kit or two competitor enzymatic fragmentation kits. High-quality DNA fragmentation times were selected to target final library sizes of approximately 500 bp per manufacturer’s published recommendations (5, 10, and 10 minutes at 37℃, respectively). Damaged DNA fragmentation times were fixed at 10 minutes at 37℃. All libraries were amplified for 6 PCR cycles. Final library distributions and yields were assessed using a D1000 assay by TapeStation (Agilent).
Figure 4B. Efficient library preparation across a wide range of sample qualities. Libraries were constructed in duplicate from 25 ng of either high-quality or formalin-compromised DNA of moderate to severe damage (HD799 and HD803, respectively; Horizon Discovery) using the Watchmaker DNA Library Prep Kit or two competitor enzymatic fragmentation kits. High-quality DNA fragmentation times were selected to target final library sizes of approximately 500 bp per manufacturer’s published recommendations (5, 10, and 10 minutes at 37℃, respectively). Damaged DNA fragmentation times were fixed at 10 minutes at 37℃. All libraries were amplified for 6 PCR cycles. Final library distributions and yields were assessed using a D1000 assay by TapeStation (Agilent).
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.

Figure 5. Library sizes are easily tailored to application-specific needs. Libraries were constructed from 50 ng of human genomic DNA. A fragmentation reaction time titration was conducted using 30°C (3, 5, and 15 minutes) and 37°C (30 minutes) incubation temperatures. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
Tunable fragmentation ensures compatibility with a broad range of applications
Figure 5. Library sizes are easily tailored to application-specific needs. Libraries were constructed from 50 ng of human genomic DNA. A fragmentation reaction time titration was conducted using 30°C (3, 5, and 15 minutes) and 37°C (30 minutes) incubation temperatures. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
Figure 5. Library sizes are easily tailored to application-specific needs. Libraries were constructed from 50 ng of human genomic DNA. A fragmentation reaction time titration was conducted using 30°C (3, 5, and 15 minutes) and 37°C (30 minutes) incubation temperatures. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).
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.

Figure 6. Even coverage of complex genomes. Libraries were prepared from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation or two widely used enzymatic fragmentation kits per manufacturer’s recommendations.
Uniform sequence coverage improves sequencing economy
Figure 6. Even coverage of complex genomes. Libraries were prepared from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation or two widely used enzymatic fragmentation kits per manufacturer’s recommendations.
Figure 6. Even coverage of complex genomes. Libraries were prepared from 1 ng of human genomic DNA using the Watchmaker DNA Library Prep Kit with Fragmentation or two widely used enzymatic fragmentation kits per manufacturer’s recommendations.
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