A new workflow for FFPE tumor samples enables a streamlined solution for structural variant detection and phasing of somatic mutations through long read sequencing

INTRODUCTION: HiFi long-read sequencing provides highly accurate reads of fragments from 500 bp to 25,000 bp in length. These long reads offer an agnostic measure of sequence length and enable structural variant detection, phasing, and direct methylation analysis. However, FFPE tissue has been difficult for HiFi sequencing because extraction often yields short, damaged fragments and fixation crosslinks DNA which disrupts long-read sequencing. METHODS: We applied the Covaris truXTRAC FFPE extraction by Adaptive Focused Acoustics (AFA) to recover long DNA fragments from FFPE blocks, including molecules up to 5000 bp. To maximize sequencing efficiency, we developed a Kinnex-based library preparation that concatenates multiple FFPE fragments into longer molecules. Kinnex involves PCR, which removes methylation information but preserves sequence quality and contiguity. The Kinnex for FFPE workflow requires 50 ng of input DNA per sample. RESULTS: Brain, kidney, and uterine tumor FFPE samples were sequenced using this approach, generating more than 100 million HiFi reads per sample with mean read lengths of 750-1,500 bp. The resulting data showed high-quality reads and consistent variant detection across the genome. Variant calling detects >11,000 structural variants and >5.1M small variants per sample, with 60% of variants phased into haplotypes. CONCLUSIONS: Combining Covaris long-fragment extraction with Kinnex library concatenation makes FFPE samples compatible with HiFi long-read sequencing. Using HiFi sequencing on samples prepared from Covaris-extracted DNA generated high yield sequencing libraries from FFPE samples across diverse tissue types demonstrating consistent performance across varying DNA quality and tissue cellularity. This workflow enables comprehensive analysis of archived tumor genomes, offering a more complete view of genomic variation and fragment characteristics from clinically relevant samples.