WATCHMAKER DNA LIBRARY PREP KITS

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Watchmaker DNA Library Prep Kits increase the conversion of template DNA into sequenceable library molecules to improve coverage and sequencing economy, especially with low-input liquid biopsy samples (cfDNA and ctDNA). The streamlined and automation-friendly workflow supports robust and flexible library preparation from fragmented DNA templates for a wide variety of sequencing applications, including WGS and targeted sequencing.

Prepare high-complexity libraries from challenging samples, such as cfDNA, FFPE, and ChIP material, with minimal bias, artifacts, and polymerase errors. Pair with Equinox® Uracil Tolerant Library Amplification Kits for methyl-sequencing applications.

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 Key Features and Benefits

  • Convert more DNA into sequenceable library molecules for enhanced sensitivity — particularly with clinically relevant cfDNA from liquid biopsies
  • Prepare highly complex libraries from as little as 500 pg (and up to 1 µg)
  • Generate libraries in under 2 hours with a simple, automatable workflow
  • Supports DNA methylation analysis when paired with Equinox Uracil Tolerant Amplification Kits for superior yield and uniformity
Non-invasive pre-natal testing: Embracing low-input methodologies

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

Applications

  • cfDNA and circulating tumor DNA (ctDNA) analysis
  • DNA methylation analysis* (Methyl-seq)
  • Chromatin immunoprecipitation sequencing (ChIP-seq)
  • Somatic mutation calling and other low-frequency variant detection assays
  • Inherited disease sequencing
  • Fragmentomics
  • cfDNA and circulating tumor DNA (ctDNA) analysis
  • DNA methylation analysis* (Methyl-seq)
  • Chromatin immunoprecipitation sequencing (ChIP-seq)
  • Somatic mutation calling and other low-frequency variant detection assays
  • Inherited disease sequencing
  • Fragmentomics
  • Human whole genome sequencing (WGS), including PCR-free
  • Whole exome sequencing (WES)
  • Microbial and metagenomic sequencing
  • Viral genome sequencing
  • Amplicon sequencing
  • Human whole genome sequencing (WGS), including PCR-free
  • Whole exome sequencing (WES)
  • Microbial and metagenomic sequencing
  • Viral genome sequencing
  • Amplicon sequencing

* In combination with Equinox Uracil Tolerant Library Amplification Kits

Workflow

Figure 1. Simple and automatable DNA library prep workflows.

Figure 1. Simple and automatable DNA library prep workflows. Both the Watchmaker DNA Library Prep Kit (required for cfDNA and DNA methylation analysis) and the Watchmaker DNA Library Prep Kit with Fragmentation (suitable for FFPE library prep and other genomics applications) enable library prep in as few as two hours and were designed with automation in mind, including generous overages.

Key Performance Data

Convert more DNA to sequenceable library for improved coverage and complexity

Improved conversion efficiency with challenging, low-input samples makes the Watchmaker DNA Library Prep Kit an excellent option for template-limited applications, such as liquid biopsy. Increased conversion of input DNA into adapter-ligated library translates to improved library complexity and coverage.

Figure 2A. Improve yields with cfDNA. TapeStation traces of final libraries. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Figure 2B. Improve yields with cfDNA. Final library yield as assessed by TapeStation analysis. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Convert more DNA to sequenceable library for improved coverage and complexity
Figure 2A. Improve yields with cfDNA. TapeStation traces of final libraries. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Figure 2A. Improve yields with cfDNA. TapeStation traces of final libraries. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Figure 2B. Improve yields with cfDNA. Final library yield as assessed by TapeStation analysis. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Figure 2B. Improve yields with cfDNA. Final library yield as assessed by TapeStation analysis. Watchmaker library yields were 9-fold higher compared to libraries prepared with other kits. No adapter-dimers were observed with any protocol. Libraries were prepared in triplicate from 1 ng of Isopure cfDNA 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.
Industry-leading performance with cell-free DNA

Naturally fragmented cell-free DNA (cfDNA) is an emerging, noninvasive biomarker for a wide range of diseases and conditions that is revolutionizing many fields of research — from oncology to prenatal and transplantation medicine. The Watchmaker DNA Library Prep Kit offers a streamlined, high-performance workflow compatible with both direct and targeted cfDNA sequencing that minimizes sample loss, adapter-dimer contamination, biases, and artifacts.

Figure 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. The shaded region indicates improved coverage of the Watchmaker libraries in comparison to other vendors. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Figure 3B. Increase coverage of liquid biopsy samples. Deduplicated UMI coverage for libraries sequenced to near-saturation (duplication rates ≥95%). Mean coverage for Watchmaker libraries was 20 – 30% higher compared to libraries prepared with other kits. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Industry-leading performance with cell-free DNA
Figure 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. The shaded region indicates improved coverage of the Watchmaker libraries in comparison to other vendors. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Figure 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. The shaded region indicates improved coverage of the Watchmaker libraries in comparison to other vendors. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Figure 3B. Increase coverage of liquid biopsy samples. Deduplicated UMI coverage for libraries sequenced to near-saturation (duplication rates ≥95%). Mean coverage for Watchmaker libraries was 20 – 30% higher compared to libraries prepared with other kits. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Figure 3B. Increase coverage of liquid biopsy samples. Deduplicated UMI coverage for libraries sequenced to near-saturation (duplication rates ≥95%). Mean coverage for Watchmaker libraries was 20 – 30% higher compared to libraries prepared with other kits. Cell-free DNA libraries were constructed in triplicate from 1 ng, 10 ng, or 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™. Data were randomly subsampled to 25 million read pairs per library.
Improve variant calling sensitivity for liquid biopsy samples

Liquid biopsy applications require the detection of low-abundance variants (typically < 1%) from very limited inputs. Increased conversion of cfDNA template into sequenceable library results in higher library complexity from limited inputs, which translates to deeper coverage and highly sensitive and reliable variant calling.

Figure 4A. High-depth coverage of rare variants. Percentage of bases covered at ≥3,000X. The Watchmaker kit offered a 3.5-fold improvement over other kits, which has significant implications for variant detection sensitivity and sequencing economy from cfDNA samples. Statistical modeling determined 3,000X to be the minimum coverage needed to call very rare variants (AF=0.1%) within a 95% confidence interval, with an alternative allele count ≥1).
Figure 4B. High-depth coverage of rare variants. Base coverage depth for the six SNVs expected at an AF of 0.1%. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. See Figure 3 for sample, library preparation, target enrichment, and sequencing information.
Improve variant calling sensitivity for liquid biopsy samples
Figure 4A. High-depth coverage of rare variants. Percentage of bases covered at ≥3,000X. The Watchmaker kit offered a 3.5-fold improvement over other kits, which has significant implications for variant detection sensitivity and sequencing economy from cfDNA samples. Statistical modeling determined 3,000X to be the minimum coverage needed to call very rare variants (AF=0.1%) within a 95% confidence interval, with an alternative allele count ≥1).
Figure 4A. High-depth coverage of rare variants. Percentage of bases covered at ≥3,000X. The Watchmaker kit offered a 3.5-fold improvement over other kits, which has significant implications for variant detection sensitivity and sequencing economy from cfDNA samples. Statistical modeling determined 3,000X to be the minimum coverage needed to call very rare variants (AF=0.1%) within a 95% confidence interval, with an alternative allele count ≥1).
Figure 4B. High-depth coverage of rare variants. Base coverage depth for the six SNVs expected at an AF of 0.1%. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. See Figure 3 for sample, library preparation, target enrichment, and sequencing information.
Figure 4B. High-depth coverage of rare variants. Base coverage depth for the six SNVs expected at an AF of 0.1%. All expected variants were detected in all replicates, except for one true positive (*), missed in one of the NEB replicate libraries. See Figure 3 for sample, library preparation, target enrichment, and sequencing information.
Increase coverage with FFPE samples

FFPE samples are ubiquitously used in NGS analysis of clinical samples, but continue to pose challenges due to highly variable quality and limited inputs. The Watchmaker DNA Library Prep Kit with Fragmentation is recommended for FFPE samples, but the standard DNA Prep Kit offers similar workflow and performance advantages for FFPE samples that are already fragmented or in settings where sonication is the preferred shearing method.

Figure 5. Improved coverage from FFPE samples of variable quality. Coverage for libraries prepared with the Watchmaker solution was 10 – 50% higher in comparison to other kits, with the most significant differences observed for low- and medium-quality samples. FFPE libraries were prepared from 10 ng or 50 ng of low- (LQ), medium- (MQ), and high-quality (HQ) FFPE samples using 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™. Data were randomly subsampled to 16 million read pairs per library.
Increase coverage with FFPE samples
Figure 5. Improved coverage from FFPE samples of variable quality. Coverage for libraries prepared with the Watchmaker solution was 10 – 50% higher in comparison to other kits, with the most significant differences observed for low- and medium-quality samples. FFPE libraries were prepared from 10 ng or 50 ng of low- (LQ), medium- (MQ), and high-quality (HQ) FFPE samples using 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™. Data were randomly subsampled to 16 million read pairs per library.
Figure 5. Improved coverage from FFPE samples of variable quality. Coverage for libraries prepared with the Watchmaker solution was 10 – 50% higher in comparison to other kits, with the most significant differences observed for low- and medium-quality samples. FFPE libraries were prepared from 10 ng or 50 ng of low- (LQ), medium- (MQ), and high-quality (HQ) FFPE samples using 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™. Data were randomly subsampled to 16 million read pairs per library.
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