Abstract 5049: Haplotype-specific copy number profiling from long reads sequencing data

Abstract Introduction. Copy number alterations (CNA) is a phenomenon during cancer progression where some regions of the affected genome may gain or lose an additional copy. This results in heterogeneous collections of cancer cells or clones, profiling and classification of these distinct classes play a vital role in understanding the cancer heterogeneity and progression to better inform diagnosis and treatment. While there are several short-reads haplotype-specific CNA profiling tools, no such long-read tool exists. Here we present Wakhan, a tool to analyze haplotype-specific chromosome-scale somatic copy number aberrations and aneuploidy using long reads (Oxford Nanopore, PacBio). Methods. Wakhan extracts Single Nucleotide Polymorphism (SNP) frequencies from tumor data using pileup statistics from BAM files based on phased heterozygous SNPs present in normal samples. Due to loss-of-heterozygosity or lower coverage in some genome regions, phasing tools break the phase-sets contiguity resulting in phase-switch errors in diploid genomes. Wakhan detects those phase switch regions and corrects them by taking into consideration the changes in haplotype-specific coverage. To detect contiguous segments with distinct copy numbers, breakpoints from structural variant callers (such as Severus) are used. For integer copy-numbers assignment and karyotype reconstruction, Wakhan performs gaussian mixture deconvolution to find the first copy number peak and subsequent peaks difference to assign copy numbers. Wakhan also detects subclonal events as segments that don’t fit the integer copy number model. Tumor purity and ploidy estimation is also detected. Wakhan generates interactive plots for overall genome coverage with customized bin size, phase blocks, SNPs frequencies and copy number segmentation, as well as integer/fractional copy numbers. Wakhan is available as open-source tool at: https://github.com/KolmogorovLab/Wakhan Results. We applied Wahkan to detect copy numbers changes resulting from breakage-fusion-bridge (BFB) events and copy-neutral loss-of-heterozygosity (LOH) events in multiple HPV-infected cell lines (CaSki, SNU1000, SCC152, HT3). In pediatric acute myeloid leukemia (AML) samples, normal karyotypes have been observed, as expected. Additionally, we analyzed several tumor/normal cell lines (COLO829, H2009, H1937, H1437 and H1954) which exhibit many copy number changes and LOH events. In COLO829, considerable aneuploidy was observed across the genome. We also compared Wakhan against short-read based copy-number profiling methods, such as HATCHet on these cancer cell lines. Overall, through long-reads we got consistent haplotype specific copy-number results as compared to Illumina data, but long-read reconstruction had better precision to smaller CNAs. In the H1437 cell line we also observed sub-clonal events in chromosomes 6 and 21. Citation Format: Tanveer Ahmad, Mikhail Kolmogorov, Ayse Keskus, Ataberk Ataberk Donmez, Midhat Farooqi. Haplotype-specific copy number profiling from long reads sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5049.