xGen cfDNA & FFPE DNA Library Prep Kit paired with UMIs helps to enable complexity in discovery
We extracted DNA from biobank sourced material which included matched FFPE-tumor, adjacent fresh-frozen normal, and plasma samples from three donors (Figure 1). The AnaPrep FFPE DNA Extraction Kit (BioChain) and the cfPure® V2 Cell-Free DNA Extraction
Kit (BioChain) were used to perform the extraction. Depending on the sample, one of several methods were used to assess its quality:
- Fluorometric quantification—Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific)
- Capillary electrophoresis—Bioanalyzer HS DNA chip (Agilent)
- qPCR—Kapa hgDNA Quantification and QC Kit (Roche)
Sequencing libraries were generated with 100 ng of DNA extracted from the FFPE samples and adjacent fresh-frozen normal samples of the three donors. Despite input sample quality, the xGen cfDNA & FFPE DNA Library Prep Kit generated high-yield libraries.
These libraries were captured in singleplex with a custom xGen pan-cancer hyb panel and sequenced. Picard was used to evaluate library preparation and hybrid capture performance including HS library size (Figure 2), duplicate rate, and coverage after
standard start-stop deduplication. Our experimental goal was to identify as many variants as possible.
High-yield libraries were generated with 25 ng of cfDNA from each of the trios and captured with subject-matched custom xGen Custom Hyb Panels. Incorporation of unique dual indexes (UDIs) ensured accuracy and prevented sample misassignment. Despite the
small size of these panels, we obtained high on-target rates and achieved a sequence depth sufficient to reach duplication rates of >80%, which is recommended for collapsed read analysis to enable error correction (Figure 3A). Collapsing reads
uses unique molecular identifiers (UMIs) to remove sample-prep, library-prep, and sequencing errors, allowing reliablevariant calling of ultra low frequency variants.
Mapped reads were used to generate collapsed single and combined read families, as outlined in the xGen cfDNA and FFPE DNA Library Prep Kit Analysis Guidelines. The combination of the xGen cfDNA
& FFPE DNA Library Prep Kit with xGen Custom Hyb Panels capture resulted in high conversion rates, complexity, and coverage for cfDNA (Figure 3B).