Cover your bases
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.
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
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
- 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 Amplification Kits
Workflow
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 a 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.
Convert more DNA to sequenceable library for improved coverage and complexity
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.
Industry-leading performance with cell-free DNA
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.
Improve variant calling sensitivity for liquid biopsy samples
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 ... MORE
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.
BROCHURES
Watchmaker DNA Library Prep Kit Brochure
Liquid Biopsy Methyl Sequencing PCR-free WGS Whole Exome Sequencing Whole Genome Sequencing Element Illumina Singular Ultima DNA Blood FFPE Microbial DNA NGS DNA Library PrepThe Watchmaker DNA Library Prep Kit enhances sequencing sensitivity, especially for low-input cfDNA from liquid biopsies, by efficiently converting DNA into sequenceable library molecules. It supports applications like cfDNA analysis, DNA methylation studies, ChIP-seq, and low-frequency variant detection, with a streamlined workflow that prepares libraries in under two hours.
TECHNICAL LITERATURE
Non-invasive prenatal testing: Embracing low-input methodologies
Non-invasive prenatal testing Illumina DNA Plasma/cfDNA DNA NGS DNA Library PrepThis case study highlights how Trisomy Test Ltd improved its NIPT workflow using the Watchmaker DNA Library Prep Kit to generate reliable results from cfDNA inputs as low as 0.5 ng. The streamlined workflow enabled faster turnaround, higher success rates in low fetal fraction samples, and efficient processing of nearly 7,000 clinical specimens.
VIDEOS
Non-invasive pre-natal testing: Embracing low-input methodologies
Non-invasive prenatal testing Whole Genome Sequencing Illumina DNA Blood Plasma/cfDNA DNA NGS DNA Library PrepAs presented at ESHG 2024, this video provides a case study on the use of the Watchmaker DNA Library Prep Kits for non-invasive prenatal testing to improve turnaround times and reduce the percentage of uninformative results.
Sequenomics: Maximizing data quality, minimizing process complexity in NGS
Illumina DNA RNA Blood FFPE Plasma/cfDNA DNA NGS RNA NGS DNA Library Prep DNA Library Prep with Fragmentation mRNA Library Prep RNA Library Prep with Polaris DepletionThis video highlights Watchmaker’s approach to “sequenomics” — the integration of biochemistry, workflow design, and automation to enhance sequencing efficiency while reducing complexity and hands-on time.
AUTOMATION RESOURCES
Streamline Watchmaker’s DNA Library Prep Kit for pre-fragmented DNA on the Sciclone G3 NGSx Workstation
Lab Automation DNA Cell Line DNA NGS DNA Library Prep RevvityThis application note details the automation of the Watchmaker DNA Library Prep Kit on the Sciclone® G3 NGSx workstation, processing up to 96 samples with flexible input and cleanup options. The workflow reduces hands-on time and minimizes errors, enhancing efficiency and reproducibility in NGS applications.
POSTERS
Correlation of pathogenic somatic mutation presence in tumor tissue and plasma-derived extracellular vesicles (FYR Diagnostics)
Liquid Biopsy Variant Calling Whole Genome Sequencing Element DNA FFPE Plasma/cfDNA DNA NGS DNA Library PrepThis poster from FYR Diagnostics explores the potential of using DNA from plasma-derived extracellular vesicles (EVs) as a minimally invasive method for detecting clinically significant somatic mutations in cancer patients. It compares somatic variants in tumor tissue with those in matched plasma EV DNA across brain, colorectal, and thyroid cancers, demonstrating that EV DNA can identify unique pathogenic variants not detected in cell-free DNA (cfDNA), highlighting its promise as a complementary tool in liquid biopsy applications.
Overcoming sample quality and quantity limitations for NGS analysis of clinically relevant samples
Liquid Biopsy Targeted Sequencing Whole Genome Sequencing DNA FFPE Plasma/cfDNA DNA NGS DNA Library Prep DNA Library Prep with FragmentationThis poster addresses challenges in next-generation sequencing (NGS) of clinically relevant samples, specifically cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) tissues, which often suffer from limited quantity and quality. It highlights how Watchmaker Genomics' innovative library preparation kits enhance scalability and sensitivity, enabling high-quality library construction from these challenging samples. These advancements facilitate more accurate and reliable NGS analyses in clinical genomics.
PROTOCOLS
Watchmaker DNA Library Prep Kit User Guide
DNA NGS DNA Library PrepProtocol for Watchmaker DNA Library Prep Kit for Fragmented Double-Stranded DNA, which supports robust and flexible library preparation from fragmented DNA templates for various sequencing applications.
| Input Range | 500 pg - 1 µg |
|---|---|
| Kit contents | ER/AT Buffer ER/AT Enzyme Ligation Buffer Ligation Enzyme Equinox® Amplification Master Mix (2X, optional) P5/P7 Primer Mix (10X, optional) |
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life | (24 rxn): ≥ 6 months (> 24 rxn): ≥ 12 months |
What are the recommended applications for the Watchmaker DNA Library Prep Kit?
The kit is designed for highly efficient conversion of variable quality DNA into Illumina-compatible sequencing-ready libraries. The workflow was developed to address the performance needs in next-generation sequencing (NGS) applications for liquid biopsies, ultra-low variant detection, bisulfite sequencing and hybrid capture. The kit is designed to support a variety of applications including the following:
- Circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) analysis
- Methyl-seq (in combination with the Equinox® Uracil Tolerant Library Amplification Kit)
- Whole-genome sequencing (including PCR-free workflows)
- Targeted sequencing protocols employing hybridization capture
- Amplicon sequencing (for post-amplification adapter ligation)
- High- and low-complexity genomes and genomes with extreme (15 - 85%) GC content
- Preparation of libraries from FFPE tissue
- Mechanically or enzymatically sheared double-stranded DNA, plasmid DNA and long PCR products
- Workflows that employ unique molecular identifiers (UMIs) for improved sensitivity
What kinds of fragmented DNA are supported by the Watchmaker DNA Library Prep Kit?
The Watchmaker DNA Library Prep Kit supports robust and flexible library preparation of DNA fragments generated by biologically occurring DNA fragmentation such as cell-free DNA (cfDNA), mechanical (e.g., Covaris®) or enzymatic fragmentation methods, as well as PCR products.
What DNA fragment length does the Watchmaker DNA Library Prep Kit support?
The Watchmaker DNA Library Prep Kit supports library preparation of fragments ranging from approximately 150 bp to 500 bp.
Is the Watchmaker DNA Library Prep Kit compatible with FFPE DNA?
The Watchmaker DNA Library Prep Kit is compatible with DNA extracted from formalin fixed paraffin-embedded tissues (FFPE). Due to the significant damage incurred by fixation and extraction, conversion efficiencies for FFPE DNA samples will most likely be lower and may be more variable sample to sample. The quality of FFPE DNA can vary greatly depending on factors such as fixation, storage, and extraction method. This variation can have a significant impact on the library preparation efficiency.
Should the DNA extracted from FFPE require fragmentation, we would recommend using our Watchmaker DNA Library Prep Kit with Fragmentation.
Is it necessary to quantitate my DNA prior to preparing libraries?
Input DNA should preferably be quantitated post-fragmentation and/or purification, immediately prior to starting the End Repair and A-tailing reactions. A fluorescence-based method such as Qubit or PicoGreen™ should preferably be used over a spectrophotometric method for accuracy.
If DNA is only quantified prior to fragmentation and/or further purification, the actual input into library preparation may be lower. This should be considered when evaluating library preparation efficiency and during library amplification cycle number optimization.
What is the recommended DNA input mass for the DNA Library Prep Kit?
The kit is compatible with a broad range of DNA input amounts (0.5 ng - 1000 ng). Conversion efficiencies should be high across this range when working with high-quality DNA inputs.
Should I really vortex the End Repair and A-tailing (ER/AT) reactions?
Yes. Vortexing the master mix components, master mix, and ER/AT reaction ensures consistent end-repair and A-tailing between samples. When preparing a small number of reactions, it is acceptable to mix by pipetting.
How should I adjust the adapter concentration for different DNA input amounts?
We recommend using different adapter concentrations dependent on the type of adapter used (stubby or full-length) as well as on the DNA type and input amount.
For ligation of stubby adapters, we recommend using an adapter concentration of 15 µM for all DNA types (including FFPE DNA and cfDNA), irrespective of DNA input amount.
For ligation of full-length adapters, we recommend using an adapter concentration of 15 µM for all input amounts of cfDNA. For non-cfDNA inputs (including FFPE DNA), we recommend using the following adapter concentrations dependent on DNA input amount:
| DNA Input (ng) | Adapter Concentration (µM) |
|---|---|
| > 50 | 15.0 |
| 25 - 50 | 7.5 |
| 10 - 24 | 3.0 |
| 2 - 9 | 1.5 |
| < 2 | 0.3 |
Adapter concentrations may need to be further optimized based on DNA input mass, quality (FFPE), fragment size, and adapter source.
We recommend using different adapter concentrations dependent on the type of adapter used (stubby or full-length) as well as on the DNA type and input amount.
For ligation of stubby adapters, we recommend using an adapter concentration of 15 µM for all DNA types (including FFPE DNA and cfDNA), irrespective of DNA input amount.
For ligation of full-length adapters, we recommend using an adapter concentration of 15 µM for all input amounts of cfDNA. For non-cfDNA inputs (including FFPE DNA), we recommend using the following adapter concentrations dependent on DNA input amount:
| DNA Input (ng) | Adapter Concentration (µM) |
|---|---|
| > 50 | 15.0 |
| 25 - 50 | 7.5 |
| 10 - 24 | 3.0 |
| 2 - 9 | 1.5 |
| < 2 | 0.3 |
Adapter concentrations may need to be further optimized based on DNA input mass, quality (FFPE), fragment size, and adapter source.
How many cycles of library amplification are recommended?
The recommended number of cycles required for amplification is dependent on the DNA input amount into library prep as well as required target library yields. See the User Guide for more specific recommendations.
Does the Watchmaker DNA Library Prep Kit support a PCR-free workflow?
Yes. The kit supports PCR-free workflows for input DNA of sufficient mass and quality. For workflows where library amplification is desirable or required, the kit includes the Equinox Amplification Master Mix.
However, libraries which are generated from low inputs and/or with truncated (stubby) adapters will require amplification prior to qPCR-based library quantification and/or sequencing.
What adapters are compatible with the Watchmaker DNA Library Prep Kit?
Any adapter with a 3' overhang T is compatible. Note that adapter quality impacts overall library preparation efficiency.
Here are some options from IDT:
- xGen Stubby Adapter and UDI Primers (Catalog number 10005976 or 10005921). This solution involves ligating a stubby or truncated Y adapter to the library during ligation, then adding in unique dual indexes with PCR primers during library amplification to incorporate sample barcodes for multiplexed sequencing.
- xGen UDI-UMI Adapters (Catalog number 10006914 and 10005903). These are full-length Y-adapters (unique dual sample barcodes, with optional UMIs) which are ligated. These can be used for PCR-free applications if sufficient input is used in library construction.
Is the Watchmaker DNA Library Prep Kit compatible with Methyl-seq?
Yes. The Watchmaker DNA Library Prep Kit can be used to prepare dU-containing DNA libraries when they are amplified with one of the Watchmaker Equinox Uracil Tolerant Amplification Kits post-ligation.
Are there any safe stopping points in the Watchmaker DNA Library Prep Kit workflow?
Yes. There is a safe stopping point after the adapter ligation SPRI cleanup step. Samples can be stored at 4°C for up to 1 week and at -20°C for up to 1 month.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling - including private label - designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.
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| Description | 24 rxn | 96 rxn | |
|---|---|---|---|
| Watchmaker DNA Library Prep Kit incl. Equinox® Library Amplification Master Mix (2X) and P5/P7 Primer Mix (10X) | 7K0102-024 | 7K0102-096 | Request a quote |
| Watchmaker DNA Library Prep Kit (PCR-free) | 7K0101-024 | 7K0101-096 | Request a quote |
| Watchmaker DNA Library Prep Kit (w/o primers); incl. Equinox Library Amplification Master Mix (2X) | 7K0103-024 | 7K0103-096 | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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