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

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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.
‭‬图 1：High-depth coverage of rare variants. 使用 25 ng 参考样品 (HD779, Horizon Discovery)（其中包含 8 个经验证的等位基因频率 (AF) 为 0.1% 的突变）构建一式三份无细胞 DNA 文库。采用 Watchmaker DNA 文库制备试剂盒、KAPA HyperPrep试剂盒或 NEBNext®Ultra™II DNA 文库制备试剂盒，按照各制造商推荐的方案制备文库。使用 37 kb 定制肿瘤面板进行十二样本靶向富集。Sequencing was performed on a NovaSeq™ 6000. 数据随机再取样至每个文库的 25 百万读取配对。所有预期的变体在所有重复中均被检测到，除了一个真阳性（*），在 NEB 重复文库中缺失。Watchmaker libraries showed higher coverage depth for all mutations.
Increase variant calling sensitivity from liquid biopsy samples
‭‬图 1：High-depth coverage of rare variants. 使用 25 ng 参考样品 (HD779, Horizon Discovery)（其中包含 8 个经验证的等位基因频率 (AF) 为 0.1% 的突变）构建一式三份无细胞 DNA 文库。采用 Watchmaker DNA 文库制备试剂盒、KAPA HyperPrep试剂盒或 NEBNext®Ultra™II DNA 文库制备试剂盒，按照各制造商推荐的方案制备文库。使用 37 kb 定制肿瘤面板进行十二样本靶向富集。Sequencing was performed on a NovaSeq™ 6000. 数据随机再取样至每个文库的 25 百万读取配对。所有预期的变体在所有重复中均被检测到，除了一个真阳性（*），在 NEB 重复文库中缺失。Watchmaker libraries showed higher coverage depth for all mutations.
Figure 1. High-depth coverage of rare variants. 使用 25 ng 参考样品 (HD779, Horizon Discovery)（其中包含 8 个经验证的等位基因频率 (AF) 为 0.1% 的突变）构建一式三份无细胞 DNA 文库。采用 Watchmaker DNA 文库制备试剂盒、KAPA HyperPrep试剂盒或 NEBNext®Ultra™II DNA 文库制备试剂盒，按照各制造商推荐的方案制备文库。使用 37 kb 定制肿瘤面板进行十二样本靶向富集。Sequencing was performed on a NovaSeq™ 6000. 数据随机再取样至每个文库的 25 百万读取配对。所有预期的变体在所有重复中均被检测到，除了一个真阳性（*），在 NEB 重复文库中缺失。Watchmaker libraries showed higher coverage depth for all mutations.
  • 由于固定过程中发生的模板损坏和残留交联的存在，处理福尔马林固定石蜡包埋样品本身就具有挑战性。
  • 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.
‭‬图 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.
‭‬图 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
‭‬图 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.
‭‬图 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.
‭‬图 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.
‭‬图 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
‭‬图 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.
‭‬图 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.
图 4. 总聚合酶错误率最多降低 40%。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
图 4. 总聚合酶错误率最多降低 40%。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.
图4，高达40%的总体聚合酶错误率的降低。 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.
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Equinox 文库扩增试剂盒

Equinox 文库扩增试剂盒

一种超高保真 DNA 聚合酶及预混反应液，以优化的热启动预混格式，专为高效、低偏好的NGS文库扩增而优化。

Watchmaker DNA 测序解决方案

Watchmaker DNA 文库制备解决方案

工作流程快，可处理所有样本类型，转化率高，偏差和伪影更少，结果可重复

Watchmaker RNA 文库制备解决方案

RNA 文库制备解决方案

具有挑战性的临床相关样品类型（例如福尔马林固定石蜡包埋和低输入）的性能，有着高度的灵敏性、可扩展性和稳健的化学特性

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