Twist cfDNA Library Preparation Kit

Yes, the engineered ligase doesn’t rely on crowding agents like PEG for maximum ligation. PEG is automation unfriendly, increases variability/viscosity, and impacts downstream bead-based purification (SPRI). We recommend 0.9X SPRI as the optimal ratio for cfDNA recovery (~150-167 bp). For shorter cfDNA fragments, we recommend increasing the SPRI ratio to 1.0X or 1.1X 

Yes, the kit’s recommended protocol allows for 35 μl of cfDNA input volume. Larger volume inputs of up to 50 μl have been tested with no negative impact on the performance of the kit. In this scenario, we recommend reducing the volume of water in the ligation reaction to compensate for the increased volume in end repair. 

No, the kit can handle a wide range of sample inputs from 0.5 ng to as high as 500 ng.

Adapter amounts and PCR cycles will need to be varied according to mass input amounts.  

Yes. The kit can be used with low input DNA samples that do not need fragmentation, or with pre-fragmented input DNA such as samples derived from FFPE material.

In tests performed with moderate DIN (3-5) FFPE curls from 4 sample sources (HG002, HG003, HG004, and Structural Multiplex Standard),  results show that the cfDNA library preparation kit can be used on FFPE material and adapted for solid tumor workflows using cfDNA Library Prep Kit and the standard hybridization v2 protocol.

Note that DNA fragmentation is still necessary even for samples with a DIN score <2. Additionally, DNA input ranges will need to be increased to accommodate the availability of DNA from FFPE extraction (~100-500 ng per 15 μM curl).

We recommend analyzing the size distribution of the input cfDNA with a gel electropherogram assay, such as Bioanalyzer High Sensitivity DNA kit (Agilent), to ensure it is free from high molecular weight (HMW) DNA that may be carried over from cfDNA extraction. HMW DNA will negatively impact the accurate quantification of cfDNA input amounts 

Yes, the kit does prepare cfDNA libraries with UMIs.

We found that library input mass into target enrichment can affect the complexity of deeply sequenced libraries and this multiplier was determined based on empirical data. This approach ensures maximum effectiveness of the enrichment process.

For more details, refer to our cfDNA Library Preparation Kit Datasheet here: https://www.twistbioscience.com/resources/technical-document/cfdna-library-prep-kit-datasheet

We recommend referencing our datasheet to determine optimal cfDNA mass input: https://www.twistbioscience.com/sites/default/files/resources/2024-05/DOC-001484cfDNALibraryPrepKitDatasetTechDocREV1singles.pdf.

The optimal amount of cfDNA input should be determined by weighing variables such as desired sensitivity at target VAF, duplexing requirements, panel content, and available sequencing resources.

The library mass used in target enrichment is calculated based on the cfDNA sample mass input into library preparation. Use the concentration of each amplified, indexed library to calculate the volume (in μl) of each library needed for hybridization when performing a singleplex or 8-plex. This information can also be found in Appendix B of the protocol here: https://www.twistbioscience.com/resources/protocol/twist-cfdna-target-enrichment-standard-hybridization-protocol

The maximum recommended library yield into target enrichment is 12.8 μg. When pooling libraries from variable starting mass inputs, each library should have the same input mass added to the pool for uniform sequencing allocation post-capture.

To calculate that mass, multiply the highest mass input by 80x. For example:

●       With an 8-plex where the highest sample mass input is 20 ng, pool 1,600 ng from each library for a total of 12.8 μg.

●       With an 8-plex where the highest sample mass input is 10 ng, pool 800 ng from each library for a total of 6.4 μg.

The cfDNA Library Preparation Kit’s workflow constructs libraries in < 3 hours.

The recommended DNA input is 1 - 20 ng of cfDNA suspended in molecular biology grade water, 10 mM Tris-HCl pH 8.0, or Buffer EB. 

Minimum recommendations for mass input going into target enrichment are based on mass input into cfDNA library preparation for singleplex samples only (see equation). This is a recommended minimum pass into capture. Increasing target enrichment mass up to 12.8 μg can be tolerated.

The cfDNA Library Preparation Kit is shipped with dry ice and is to be stored between -25°C and -15°C upon receipt.

The cfDNA Library Preparation Kit is shipped with dry ice and is to be stored between -25°C and -15°C upon receipt.

The kit can prepare cfDNA libraries both with universal adapters for PCR with UDI primers and also with full-length UDI adapters for PCR-free and WGS applications (see cfDNA full-length UDI protocol here:

https://www.twistbioscience.com/resources/protocol/twist-cfdna-library-preparation-kit-twist-full-length-udi-adapters).

Refer to https://www.twistbioscience.com/products/ngs/library-preparation/universal-adapter-system for more information.

Additionally, the kit’s molecular biology should work with adapters and primers from other vendors. However, the adapter must contain a 3’ T overhang to facilitate the A/T ligation with the 3’ A overhang in the library molecules. 

Final library yield may be slightly lower when not using the Twist-optimized solution.

We recommend lower plexing to prevent exceeding the 12.8 μg limit. 

When stored and handled as recommended, the product will retain full performance until its listed expiry date. The product has been tested with up to eight freeze/thaw cycles, and shown no impact to expected performance.

The cfDNA Library Preparation Kit constructs indexed libraries that can be used in whole genome sequencing (WGS) experiments or downstream with Twist target enrichment kits and sequenced on Illumina next-generation sequencing (NGS) systems.

The cfDNA target enrichment kit supports a 1-plex (single) or 8-plex hybridization capture.

We see a decrease in performance based on target capture metrics. This reduced performance is related to increases in off-target. 

Overamplification can result if too many PCR cycles are used. Once PCR primers are depleted, library fragments may be single stranded or form heteroduplexes that appear as high molecular weight fragments in capillary gel electrophoresis instruments (Figure 3). These peaks are different from the di-nucleosomal peaks that are expected from cfDNA as seen in early PCR cycles. We recommend starting with the number of PCR cycles recommended in the protocol (https://www.twistbioscience.com/resources/protocol/twist-cfdna-library-preparation-twist-umi-adapter-system) and reducing PCR cycles if overamplification is observed.