Hybridization capture of FFPE DNA and cfDNA samples
We extracted DNA from matched trios of biobank sourced material from three individuals which included adjacent fresh-frozen normal tissue, matched formalin-fixed paraffin-embedded- (FFPE)-tumor tissue, and plasma samples (Figure 1). The AnaPrep FFPE DNA
Extraction Kit (BioChain) and the cfPure® V2 Cell-Free DNA Extraction Kit (BioChain) were used to perform DNA extraction. Depending on the sample, one of the following methods was 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. Despite low-input sample quality, the xGen cfDNA & FFPE DNA Library Prep Kit generated high-yield libraries.
The resultant libraries were captured in single-plex with a custom xGen Pan-Cancer hybridization panel and sequenced. Picard (Broad Institute) was used to evaluate library preparation and hybrid capture results including hybrid-selection (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 isolated from the matched plasma from each of the three individuals and captured with subject-matched custom xGen Custom Hyb Panels. Incorporation of unique dual indexes (UDIs) increased resolution and prevented sample misassignment. Despite the small size of these panels, we obtained high on-target rates and 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, which leads to reliable variant calling of ultra-low frequency variants.
Mapped reads were used to generate
collapsed single 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 complexity (Figure 3A) and coverage for cfDNA (Figure 3B).