What are the recommended applications for the Watchmaker DNA Library Prep Kit?
- 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)
- 采用杂交捕获的靶向测序方案
- 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
- 采用独特分子标签 (UMI) 提高灵敏度的工作流程
What kinds of fragmented DNA are supported by the Watchmaker DNA Library Prep Kit?
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?
是。Vortexing the master mix components, master mix, and ER/AT reaction ensures consistent end-repair and A-tailing between samples. 当制备少量反应时，可以通过移液管混合。
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 起始量 (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?
是。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?
任何带有 3' 悬垂 T 的接头都是兼容的。请注意，接头质量会影响文库的整体制备效率。
以下是 IDT 的一些选项：
- xGen 短型接头和 UDI 引物（目录号 10005976 或 10005921）。这种解决方案包括在连接过程中将一个短型或截短的 Y 型接头连接到文库，然后在文库扩增过程中添加带有 PCR 引物的独特双端索引，以引入样品标签用于多重测序。
- xGen UDI-UMI 接头（目录号 10006914 和 10005903）。这些是全长 Y 形接头（独特的双端样品条形码，带有可选的独特分子标签），可以连接。如果在文库构建中起始量足够，则这些可用于 PCR-Free 的应用。
Is the Watchmaker DNA Library Prep Kit compatible with Methyl-seq?
是。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?
是。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. 我们的证书被授予设计、开发、制造、合同制造和支持在医学研究中的基因组学应用的高性能试剂。Download the certificate here.
