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.
Highlights
- Increase variant calling sensitivity form liquid biopsy samples
- Access more information from FFPE samples
- Accurate PCR-free whole genome sequencing (WGS) without sonication
- Enable rare mutation detection with ultra-high-fidelity library amplification
- For assay developers - make realizing your next product easier
Watch now: Non-invasive Prenatal Testing: Embracing Low Input Methodologies
As presented at ESHG 2024
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 ... MORE
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.
- 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.
Access more information from 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.
Accurate PCR-free whole genome sequencing (WGS) without a Covaris
- 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 ... MORE
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.
TECHNICAL LITERATURE
Enzymatic fragmentation enables scalable preparation of high-quality FFPE libraries with minimal artifacts
Targeted Sequencing Illumina DNA Cell Line FFPE DNA NGS DNA Library Prep with FragmentationThis application note highlights the use of the Watchmaker DNA Library Prep Kit with Fragmentation for FFPE samples, offering enzymatic fragmentation that reduces artifacts and enhances sequencing data quality. The streamlined protocol minimizes sample loss and improves efficiency compared to traditional sonication methods.
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
Customizing Watchmaker DNA PCR-free Library Prep Kit for improved NovaSeq X performance
PCR-free WGS Whole Genome Sequencing Illumina DNA DNA NGS DNA Library Prep with FragmentationAs presented at ESHG 2024, this video provides recommendations and commentary on whole genome sequencing on the NovaSeq X platform.
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.
Fusion detection in pediatric oncology: Insights from transcriptome sequencing
Fusion Detection Novel Transcript Discovery Whole Transcriptome/Total RNA Sequencing Illumina FFPE RNA NGS RNA Library Prep with Polaris DepletionAs presented at AMP 2024, hear Katie Miller, PhD from Nationwide Children’s Hospital present on the value of whole transcriptome sequencing (WTS) for fusion detection, including: the benefits of WTS over DNA sequencing and targeted RNA sequencing; the importance of a workflow that can handle low inputs and damaged FFPE samples while providing robust inter-user performance; and the potential utility of gene expression information in addition to fusion calling.
POSTERS
Increasing complexity and quality of total RNA sequencing at scale (Broad Clinical Labs)
Gene Expression Analysis Lab Automation mRNA Sequencing Whole Transcriptome/Total RNA Sequencing Illumina RNA Blood RNA NGS RNA Library Prep with Polaris Depletion AgilentThis poster from Broad Clinical Labs presents a comparison between the Watchmaker RNA Library Prep with Polaris® Depletion and a standard poly(A) mRNA capture with globin depletion method using whole blood samples. The study demonstrates that the Watchmaker workflow yields more complex libraries, identifying 15% more transcripts and 3% more protein-coding genes than the standard method. Additionally, the automated Watchmaker workflow significantly reduces hands-on processing time, with a single lab user capable of generating approximately 960 libraries per week using two machines.
Cost-effective, high-resolution whole transcriptome sequencing for gene fusion detection applications (Ultima Genomics)
Fusion Detection Gene Expression Analysis Variant Calling Whole Transcriptome/Total RNA Sequencing Ultima RNA RNA NGS RNA Library Prep with Polaris DepletionThis poster presents the optimization of the Watchmaker RNA Library Prep Kit with Polaris® Depletion for use with Ultima Genomics' high-capacity sequencing platform, enabling cost-effective whole transcriptome sequencing (WTS) for gene fusion detection.
Optimized RNA-seq library preparation for fusion calling from FFPE samples
Fusion Detection Gene Expression Analysis Targeted Sequencing Whole Transcriptome/Total RNA Sequencing Illumina RNA FFPE RNA NGS RNA Library Prep with Polaris DepletionThis poster explores the use of whole transcriptome analysis (WTA) for detecting gene fusions in formalin-fixed, paraffin-embedded (FFPE) samples, comparing it to targeted enrichment methods. The study demonstrates that WTA can identify known fusions and, with optimized library preparation for longer insert sizes, facilitate de novo fusion detection. Additionally, the research shows that WTA enables gene expression analysis and fusion identification from a single dataset, offering a comprehensive approach to transcriptomic profiling in challenging FFPE samples.
A comparative analysis of library preparation technologies for RNA sequencing from FFPE samples
Gene Expression Analysis Whole Genome Sequencing Illumina RNA FFPE Frozen Tissue RNA NGS RNA Library Prep with Polaris DepletionThis poster compares RNA sequencing solutions for FFPE samples, highlighting that the Watchmaker RNA Library Prep Kit with Polaris® Depletion provides superior gene detection and reproducibility, with minimal data distortion.
An evaluation of enzymatic fragmentation in the library preparation workflow: The Watchmaker Genomics Kit (Broad Institute)
Lab Automation PCR-free WGS SNV/SNP/InDel Detection Variant Calling Whole Genome Sequencing Illumina DNA Cell Line DNA NGS DNA Library Prep with Fragmentation AgilentThis poster from the Broad Institute evaluates the Watchmaker DNA Library Prep Kit with Fragmentation, focusing on its enzymatic fragmentation method as an alternative to traditional mechanical shearing in whole genome sequencing (WGS). The study demonstrates that the enzymatic approach offers comparable sequencing metrics. The workflow is user-friendly, completing high-quality library construction in under two hours, and is suitable for scaling in throughput.
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.
Optimized enzymatic fragmentation workflow for the rapid construction of high-quality whole genome and whole exome libraries that is compatible with several blood collection methodologies
PCR-free WGS Whole Genome Sequencing Illumina DNA Blood Dried Blood Spot Saliva/buccal DNA NGS DNA Library Prep with FragmentationThis poster presents the Watchmaker DNA Library Prep Kit with Fragmentation, optimized for rapid, high-quality library construction from various DNA collection methods, including whole blood, buccal swabs, at-home blood collection devices (Tasso™), and dried blood spots. The workflow achieves high library conversion rates across sample types, with consistent sequencing performance and variant calling concordance. Adjusting post-ligation SPRI cleanup can further enhance performance, and tuning post-ligation size selection allows for larger insert libraries.
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.
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