WATCHMAKER DNA LIBRARY PREP KITS: FAQS

• 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

The Watchmaker DNA Library Prep Kit supports library preparation of fragments ranging from approximately 150 bp to 500 bp.

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.

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.

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.

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.

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:

Adapter concentrations may need to be further optimized based on DNA input mass, quality (FFPE), fragment size, and adapter source.

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.

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.

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.

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.

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.

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.

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.