Watchmaker DNA 文库制备试剂盒

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Cover your bases

Watchmaker DNA 文库制备试剂盒增加了模板 DNA 向可测序文库分子的转化，以改进覆盖率和测序经济性，特别是低输入液体活检样品（cfDNA 和 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.

使用具有挑战性的样品制备高复杂度的文库，如循环游离 DNA、福尔马林固定石蜡包埋和 ChIP 材料，偏差、伪影和聚合酶错误最少。配对 Equinox® Uracil Tolerant Library Amplification Kits 用于甲基测序应用。

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 主要特点和优点

  • 将更多的 DNA 转化为可测序的文库分子，以提高灵敏度——特别是来自液体活检的临床相关循环游离 DNA
  • 使用低至 500pg（最高 1µg）制备高度复杂的文库
  • 在 2 个小时内通过一个简单的、可自动化的工作流程生成文库
  • 当与Equinox 尿嘧啶耐受扩增试剂盒配对时，支持 DNA 甲基化分析，以获得更高的产量和均匀性
Non-invasive pre-natal testing: Embracing low-input methodologies

现在观看：Non-invasive Prenatal Testing: Embracing Low Input Methodologies
As presented at ESHG 2024

应用

  • 循环游离 DNA 和循环肿瘤 DNA (ctDNA) 分析
  • DNA 甲基化分析*（甲基测序）
  • 染色质免疫共沉淀测序（ChIP 测序）
  • 体细胞突变调用和其他低频变异检测分析
  • 遗传病测序
  • 片段化
  • 循环游离 DNA 和循环肿瘤 DNA (ctDNA) 分析
  • DNA 甲基化分析*（甲基测序）
  • 染色质免疫共沉淀测序（ChIP 测序）
  • 体细胞突变调用和其他低频变异检测分析
  • 遗传病测序
  • 片段化
  • Human whole genome sequencing (WGS), including PCR-free
  • 全外显子组测序 (WES)
  • 微生物和宏基因组测序
  • 病毒基因组测序
  • 扩增子测序
  • Human whole genome sequencing (WGS), including PCR-free
  • 全外显子组测序 (WES)
  • 微生物和宏基因组测序
  • 病毒基因组测序
  • 扩增子测序

* 与 Equinox 尿嘧啶耐受文库扩增试剂盒结合

工作流程

‭‬图 1：简单和自动化的 DNA 文库制备工作流程。

‭‬图 1：简单和自动化的 DNA 文库制备工作流程。 Watchmaker DNA 文库制备试剂盒（循环游离 DNA 和 DNA 甲基化分析需要） Watchmaker 酶法片段化 DNA 文库制备试剂盒 （适用于福尔马林固定石蜡包埋文库制备和其他基因组学应用）可以在短短两个小时内完成文库制备，并且在设计时考虑了自动化，超量投料充足。

关键性能数据

将更多的 DNA 转换为可测序文库，以改进覆盖度和复杂度

具有挑战性的低输入样品改进了转换效率，使 Watchmaker DNA 文库制备试剂盒成为模板有限应用（如液体活检）的绝佳选择。将输入 DNA 转化为接头连接文库的转化率提高，进一步改进文库复杂度和覆盖度。

‭‬图 2A. Improve yields with cfDNA. TapeStation traces of final libraries. Watchmaker 文库的产量比用其他试剂盒制备的文库高 9 倍。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.
‭‬图 2B. Improve yields with cfDNA. Final library yield as assessed by TapeStation analysis. Watchmaker 文库的产量比用其他试剂盒制备的文库高 9 倍。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.
将更多的 DNA 转换为可测序文库，以改进覆盖度和复杂度
‭‬图 2A. Improve yields with cfDNA. TapeStation traces of final libraries. Watchmaker 文库的产量比用其他试剂盒制备的文库高 9 倍。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 文库的产量比用其他试剂盒制备的文库高 9 倍。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.
‭‬图 2B. Improve yields with cfDNA. Final library yield as assessed by TapeStation analysis. Watchmaker 文库的产量比用其他试剂盒制备的文库高 9 倍。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 文库的产量比用其他试剂盒制备的文库高 9 倍。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.
在无细胞 DNA 领域具有领先行业水平的性能

自然片段化无细胞 DNA（循环游离 DNA）是一种新兴的、无创的生物标志物，用于广泛的疾病和病症，正在彻底改变许多研究领域——从肿瘤学到产前和移植医学。Watchmaker DNA 文库制备试剂盒提供了一个精简的，高性能的工作流程，它兼容直接和靶向的循环游离 DNA 测序，最大限度地减少样品损失，接头二聚体污染、偏差和伪影。

‭‬图 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. 阴影区域表示与其他供应商相比，Watchmaker 文库的覆盖度有所提高。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
‭‬图 3B. Increase coverage of liquid biopsy samples. 对于测序至接近饱和的文库进行去重的 UMI 覆盖度（重复率 ≥95％）。Watchmaker 文库的平均覆盖度比使用其他试剂盒制备的文库高 20–30%。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
在无细胞 DNA 领域具有领先行业水平的性能
‭‬图 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. 阴影区域表示与其他供应商相比，Watchmaker 文库的覆盖度有所提高。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
Figure 3A. Increase coverage of liquid biopsy samples. Cumulative coverage across the entire target region for 25 ng libraries. 阴影区域表示与其他供应商相比，Watchmaker 文库的覆盖度有所提高。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
‭‬图 3B. Increase coverage of liquid biopsy samples. 对于测序至接近饱和的文库进行去重的 UMI 覆盖度（重复率 ≥95％）。Watchmaker 文库的平均覆盖度比使用其他试剂盒制备的文库高 20–30%。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
Figure 3B. Increase coverage of liquid biopsy samples. 对于测序至接近饱和的文库进行去重的 UMI 覆盖度（重复率 ≥95％）。Watchmaker 文库的平均覆盖度比使用其他试剂盒制备的文库高 20–30%。使用 1 ng、10 ng 或 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。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 25 百万读取配对。
提高液体活检样品的变体调用灵敏度

液体活检应用需要从非常有限的输入中检测低丰度变体（通常 < 1%）。增加将循环游离 DNA 模板转换为可测序文库的过程，可以从有限的输入中获得更高的文库复杂度，从而转化为更深入的覆盖度和高度敏感和可靠的变体调用。

‭‬图 4A. High-depth coverage of rare variants. ≥3000 倍覆盖的碱基百分比。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).
‭‬图 4B. High-depth coverage of rare variants. Base coverage depth for the six SNVs expected at an AF of 0.1%. 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。See Figure 3 for sample, library preparation, target enrichment, and sequencing information.
提高液体活检样品的变体调用灵敏度
‭‬图 4A. High-depth coverage of rare variants. ≥3000 倍覆盖的碱基百分比。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. ≥3000 倍覆盖的碱基百分比。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).
‭‬图 4B. High-depth coverage of rare variants. Base coverage depth for the six SNVs expected at an AF of 0.1%. 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。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%. 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。See Figure 3 for sample, library preparation, target enrichment, and sequencing information.
增加福尔马林固定石蜡包埋样品的覆盖度

FFPE 样品普遍用于临床样品的新一代测序分析，但由于质量高度可变和输入有限，仍然构成挑战。超高 Watchmaker 酶法片段化 DNA 文库制备试剂盒 推荐用于福尔马林固定石蜡包埋样品，但标准 DNA 制备试剂盒为已经片段化的福尔马林固定石蜡包埋样品或在超声处理作为首选剪切方法的设置中提供类似的工作流程和性能优势。

图 5. 提高了可变质量福尔马林固定石蜡包埋样品的覆盖度。与其他试剂盒相比，使用 Watchmaker 溶液制备的文库的覆盖度高 10 – 50%，在低质量和中等质量样品中观察到最显著的差异。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. 使用 37 kb 定制肿瘤面板进行十二样本靶向富集。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 16 百万读取配对。
增加福尔马林固定石蜡包埋样品的覆盖度
图 5. 提高了可变质量福尔马林固定石蜡包埋样品的覆盖度。与其他试剂盒相比，使用 Watchmaker 溶液制备的文库的覆盖度高 10 – 50%，在低质量和中等质量样品中观察到最显著的差异。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. 使用 37 kb 定制肿瘤面板进行十二样本靶向富集。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 16 百万读取配对。
图 5. 提高了可变质量福尔马林固定石蜡包埋样品的覆盖度。 与其他试剂盒相比，使用 Watchmaker 溶液制备的文库的覆盖度高 10 – 50%，在低质量和中等质量样品中观察到最显著的差异。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. 使用 37 kb 定制肿瘤面板进行十二样本靶向富集。在 NovaSeq™ 上进行测序。数据随机再取样至每个文库的 16 百万读取配对。
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