Personalized reference genome-based pipeline reveals comprehensive haplotype-resolved views of cancer genomes
Cancer genome analysis relies on standard human reference genomes, but detecting somatic alterations in highly repetitive or individual-specific regions remains challenging. We developed the Personalized Reference genome-based Cancer Genome Analysis Pipeline (PRCGAP, https://github.com/yos-sk/PRCGAP), to our knowledge, the first comprehensive pipeline integrating haplotype-resolved analyses of somatic point mutations, structural variants, copy number, and DNA methylation on personalized diploid reference genomes, with each variant phased and annotated by genomic features. We applied PRCGAP to eight tumor-normal cell line pairs and three newly collected pediatric B-cell acute lymphoblastic leukemia (B-ALL) clinical samples. PRCGAP recovered most standard reference-based variants (86.7-96.4% across variant types), validated by orthogonal short-read sequencing, while additionally detecting variants that standard references missed (23.4% of SNVs and 32.6% of SVs). Notably, PRCGAP uncovered variants in centromeric and telomeric regions, and somatic single-nucleotide variants were enriched in centromere dip regions, the putative kinetochore formation sites. Using PRCGAP outputs, we identified L1 retrotransposition source sites absent from standard references and showed that a B-ALL IGH::DUX4 fusion arose from a DUX4 pseudogene within an internal D4Z4 repeat unit, rather than the canonical full-length DUX4 gene. Within a single haplotype-resolved framework, PRCGAP opens regions of the cancer genome that are inaccessible to standard references.