Solutions for Translational and Clinical Researchers

Addressing sensitivity bottlenecks in clinically relevant sequencing applications

Sensitive applications require precision tools

Clinically relevant and other highly sensitive sequencing applications require solutions that maximize the information output from samples where template mass and quality are limited. Watchmaker leverages novel enzyme engineering with multidimensional reaction optimization to develop DNA and RNA NGS library preparation solutions that better maintain data integrity – minimizing bias and improving sequence accuracy to ultimately increase sensitivity.

The resulting solutions deliver meaningful data quality improvements compared to existing commercially available products. This is especially evident when working with challenging cfDNA from liquid biopsies, FFPE, and other low-input or degraded samples. These advancements enable researchers to address more clinically relevant samples using simple, fast, and easily automated workflows.

Non-invasive pre-natal testing: Embracing low-input methodologies

Watch now: Non-invasive Prenatal Testing: Embracing Low Input Methodologies
As presented at ESHG 2024

Highlights

Sensitive solutions for clinically relevant applications

  • Liquid biopsy applications require the detection of low-abundance variants (typically <1%) from very limited inputs.
  • The Watchmaker DNA Library Prep Kit increases the conversion of cell-free DNA (cfDNA) template into sequenceable library.
  • This results in higher library complexity from limited inputs, which translates to deeper coverage and highly sensitive and reliable variant calling from cfDNA samples.
Figure 1. High-depth coverage of rare variants. Cell-free DNA libraries were constructed in triplicate from 25 ng of a reference sample (HD779, Horizon Discovery) containing eight mutations at a verified allele frequency (AF) of 0.1%. Libraries were prepared with the Watchmaker DNA Library Prep Kit, KAPA HyperPrep Kit, or NEBNext® Ultra™ II for DNA Library Prep according to each manufacturer’s recommended protocol. Twelve-plex target enrichment was performed using a 37 kb custom oncology panel. Sequencing was performed on a NovaSeq™ 6000. Data were randomly subsampled to 25 million read pairs per library. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. Watchmaker libraries showed higher coverage depth for all mutations.
Increase variant calling sensitivity from liquid biopsy samples
Figure 1. High-depth coverage of rare variants. Cell-free DNA libraries were constructed in triplicate from 25 ng of a reference sample (HD779, Horizon Discovery) containing eight mutations at a verified allele frequency (AF) of 0.1%. Libraries were prepared with the Watchmaker DNA Library Prep Kit, KAPA HyperPrep Kit, or NEBNext® Ultra™ II for DNA Library Prep according to each manufacturer’s recommended protocol. Twelve-plex target enrichment was performed using a 37 kb custom oncology panel. Sequencing was performed on a NovaSeq™ 6000. Data were randomly subsampled to 25 million read pairs per library. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. Watchmaker libraries showed higher coverage depth for all mutations.
Figure 1. High-depth coverage of rare variants. Cell-free DNA libraries were constructed in triplicate from 25 ng of a reference sample (HD779, Horizon Discovery) containing eight mutations at a verified allele frequency (AF) of 0.1%. Libraries were prepared with the Watchmaker DNA Library Prep Kit, KAPA HyperPrep Kit, or NEBNext® Ultra™ II for DNA Library Prep according to each manufacturer’s recommended protocol. Twelve-plex target enrichment was performed using a 37 kb custom oncology panel. Sequencing was performed on a NovaSeq™ 6000. Data were randomly subsampled to 25 million read pairs per library. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. Watchmaker libraries showed higher coverage depth for all mutations.
  • Processing FFPE samples is inherently challenging due to the template damage incurred during fixation and the presence of residual cross-links.
  • The Watchmaker RNA Library Prep Kit with Polaris Depletion was designed for FFPE samples and includes a novel FFPE treatment step and a reverse transcriptase specifically engineered for RNA-seq applications.
  • The result is a solution that delivers excellent sensitivity, enabling researchers to address a broader range of clinically relevant samples effectively.
Figure 2A. Access more information from FFPE. Libraries were prepared in duplicate from 10 ng of a matched fresh frozen and FFPE sample set using Watchmaker RNA Library Prep with Polaris Depletion, KAPA RNA HyperPrep with RiboErase (HMR) Globin, NEBNext Ultra II Directional RNA Library Prep with Globin & rRNA Depletion, and Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. Data were downsampled to 11M paired reads per library. Unique genes were identified using featureCounts with a cutoff of 5 deduplicated raw reads. Only genes identified in both technical replicates were counted. In a gene detection overlap analysis between the fresh frozen and FFPE data sets for each chemistry, Watchmaker FFPE libraries detect a significantly higher percentage of the unique genes identified in the fresh frozen control.
Figure 2B. Access more information from FFPE. Watchmaker detects more unique genes for both the fresh frozen and FFPE samples.
Access more information from FFPE samples
Figure 2A. Access more information from FFPE. Libraries were prepared in duplicate from 10 ng of a matched fresh frozen and FFPE sample set using Watchmaker RNA Library Prep with Polaris Depletion, KAPA RNA HyperPrep with RiboErase (HMR) Globin, NEBNext Ultra II Directional RNA Library Prep with Globin & rRNA Depletion, and Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. Data were downsampled to 11M paired reads per library. Unique genes were identified using featureCounts with a cutoff of 5 deduplicated raw reads. Only genes identified in both technical replicates were counted. In a gene detection overlap analysis between the fresh frozen and FFPE data sets for each chemistry, Watchmaker FFPE libraries detect a significantly higher percentage of the unique genes identified in the fresh frozen control.
Figure 2A. Access more information from FFPE. Libraries were prepared in duplicate from 10 ng of a matched fresh frozen and FFPE sample set using Watchmaker RNA Library Prep with Polaris Depletion, KAPA RNA HyperPrep with RiboErase (HMR) Globin, NEBNext Ultra II Directional RNA Library Prep with Globin & rRNA Depletion, and Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. Data were downsampled to 11M paired reads per library. Unique genes were identified using featureCounts with a cutoff of 5 deduplicated raw reads. Only genes identified in both technical replicates were counted. In a gene detection overlap analysis between the fresh frozen and FFPE data sets for each chemistry, Watchmaker FFPE libraries detect a significantly higher percentage of the unique genes identified in the fresh frozen control.
Figure 2B. Access more information from FFPE. Watchmaker detects more unique genes for both the fresh frozen and FFPE samples.
Figure 2B. Access more information from FFPE. Watchmaker detects more unique genes for both the fresh frozen and FFPE samples.
  • PCR-free WGS provides comprehensive genomic information with minimal bias, making it valuable for research, diagnostics, and precision medicine.
  • The Watchmaker DNA Library Prep Kit with Fragmentation delivers high library yields without the need for amplification, increased coverage across challenging promoter regions, robust variant calling, and excellent sequence coverage.
  • Many of these benefits surpass the ‘gold-standard’ method of mechanical fragmentation.
Figure 3A. Efficient conversion delivers high library yields without amplification. PCR-free WGS libraries were constructed using a variety of commercially available solutions: KAPA HyperPrep (sonication control), Watchmaker DNA Library Prep with Fragmentation, KAPA HyperPlus, KAPA EvoPlus, NEBNext Ultra II FS DNA Library Prep, and Illumina DNA PCR-Free Prep. Either 300 ng (all evaluated kits) or 75 ng (Watchmaker only) of NA12878 human genomic DNA was used as input. Workflows were optimized to deliver similarly sized final libraries by tuning fragmentation and post-ligation cleanup parameters to ensure fair and accurate comparison of key sequencing performance metrics. Watchmaker library prep yields exceeded other sonication and enzymatic fragmentation preps.
Figure 3B. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to mean coverage of challenging GC-rich promoters. The Watchmaker solution delivered libraries with the highest mean coverage of challenging promoters, even when only 75 ng of DNA was used for library prep, in comparison to other solutions, including sonication.
Figure 3C.Sensitive and precise variant calling. With respect to SNP and Indel F1-scores, the Watchmaker libraries deliver variant calling performance on-par with the sonication control.
Accurate PCR-free whole genome sequencing (WGS) without a Covaris
Figure 3A. Efficient conversion delivers high library yields without amplification. PCR-free WGS libraries were constructed using a variety of commercially available solutions: KAPA HyperPrep (sonication control), Watchmaker DNA Library Prep with Fragmentation, KAPA HyperPlus, KAPA EvoPlus, NEBNext Ultra II FS DNA Library Prep, and Illumina DNA PCR-Free Prep. Either 300 ng (all evaluated kits) or 75 ng (Watchmaker only) of NA12878 human genomic DNA was used as input. Workflows were optimized to deliver similarly sized final libraries by tuning fragmentation and post-ligation cleanup parameters to ensure fair and accurate comparison of key sequencing performance metrics. Watchmaker library prep yields exceeded other sonication and enzymatic fragmentation preps.
Figure 3A. Efficient conversion delivers high library yields without amplification. PCR-free WGS libraries were constructed using a variety of commercially available solutions: KAPA HyperPrep (sonication control), Watchmaker DNA Library Prep with Fragmentation, KAPA HyperPlus, KAPA EvoPlus, NEBNext Ultra II FS DNA Library Prep, and Illumina DNA PCR-Free Prep. Either 300 ng (all evaluated kits) or 75 ng (Watchmaker only) of NA12878 human genomic DNA was used as input. Workflows were optimized to deliver similarly sized final libraries by tuning fragmentation and post-ligation cleanup parameters to ensure fair and accurate comparison of key sequencing performance metrics. Watchmaker library prep yields exceeded other sonication and enzymatic fragmentation preps.
Figure 3B. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to mean coverage of challenging GC-rich promoters. The Watchmaker solution delivered libraries with the highest mean coverage of challenging promoters, even when only 75 ng of DNA was used for library prep, in comparison to other solutions, including sonication.
Figure 3B. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to mean coverage of challenging GC-rich promoters. The Watchmaker solution delivered libraries with the highest mean coverage of challenging promoters, even when only 75 ng of DNA was used for library prep, in comparison to other solutions, including sonication.
Figure 3C.Sensitive and precise variant calling. With respect to SNP and Indel F1-scores, the Watchmaker libraries deliver variant calling performance on-par with the sonication control.
Figure 3C.Sensitive and precise variant calling. With respect to SNP and Indel F1-scores, the Watchmaker libraries deliver variant calling performance on-par with the sonication control.
  • Sensitive variant detection requires the use of a library amplification polymerase that minimizes PCR errors to reduce false calls.
  • Equinox® is a proprietary proofreading polymerase, capable of a 40% reduction in overall polymerase error rate in comparison to KAPA HiFi HotStart ReadyMix.
  • The reduction in C>T substitutions is particularly notable, as this mutation is one of the most common mutation types in cancers.
Figure 4. Up to 40% reduction in overall polymerase error rate. Error rates were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. The Equinox Library Amplification Kit displayed a 40% reduction in overall polymerase error rate in comparison to KAPA HiFi HotStart ReadyMix.
Enable rare mutation detection with ultra-high-fidelity library amplification
Figure 4. Up to 40% reduction in overall polymerase error rate. Error rates were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. The Equinox Library Amplification Kit displayed a 40% reduction in overall polymerase error rate in comparison to KAPA HiFi HotStart ReadyMix.
Figure 4. Up to 40% reduction in overall polymerase error rate. Error rates were measured after >9 million base incorporation events in three separate reactions, using a proprietary NGS-based assay. The Equinox Library Amplification Kit displayed a 40% reduction in overall polymerase error rate in comparison to KAPA HiFi HotStart ReadyMix.
For assay developers – make realizing your next product easier
  • Navigate the complexities of productization with an experienced team who’s as committed to your success as you are
  • All aspects of our customization process are designed to serve you with speed, agility, and above all else, a commitment to quality
  • Tailored fill volumes, labeling (including white and private label), and packaging designed to your specifications
  • All products are manufactured within an ISO 13485:2016-certified QMS (download our certificate)
Discover more with Watchmaker
EQUINOX LIBRARY AMPLIFICATION KIT

Equinox Library Amplification Kits

An ultra-high-fidelity DNA polymerase in an optimized hot start master mix format, specifically optimized for high-efficiency, low-bias NGS library amplification

Watchmaker DNA-Seq Solutions

Watchmaker DNA Library Prep Solutions

Rapid workflows to address all sample types and yield high conversion rates, reduced bias and artifacts, and reproducible results

Watchmaker RNA Library Prep Solutions

RNA Library Prep Solutions

Highly sensitive, scalable, and robust chemistry with respect to performance with challenging clinically relevant sample types, such as FFPE and low inputs

REQUEST A QUOTE OR SAMPLE NOW!