Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

Area of Science:

• Genomics
• Cancer Research
• Bioinformatics

Background:

• Fresh Frozen (FF) DNA is not routinely collected in clinical settings.
• Formalin Fixed Paraffin Embedded (FFPE) tumor samples are widely available.
• FFPE samples could significantly expand cancer cohort studies if artefacts are managed.

Purpose of the Study:

• To assess the feasibility of using FFPE samples for whole genome cancer cohort studies.
• To compare somatic single nucleotide variants (SNVs) called from matching FF and FFPE samples.
• To develop and validate a variant calling strategy for FFPE material.

Main Methods:

• Somatic variants were called on matching FF and FFPE whole genome sequencing (WGS) samples using four callers (Strelka2, Mutect2, VarScan2, Shimmer).
• A heuristic approach was developed to maximize overlap (sensitivity and precision) between FF and FFPE variant calls.
• The approach was validated on nine additional matched samples and intra-tumor heterogeneity (ITH) was assessed.

Main Results:

• An average of 50% of somatic SNVs from FF samples were recovered in matching FFPE samples, with significant caller-dependent variation.
• The developed heuristic improved both precision and sensitivity, achieving an average F1-score of 0.58 across nine validation samples.
• A portion of the discrepancy between FF and FFPE samples was attributed to intra-tumor heterogeneity (ITH).

Conclusions:

• Somatic SNVs can be recovered from FFPE samples with high precision and sensitivity using an optimized variant calling strategy.
• FFPE-derived WGS data is suitable for large-scale cancer cohort studies.
• This approach maximizes the utility of archived clinical tumor samples for genomic research.