Abstract
<jats:p>Single-cell DNA sequencing (scDNA-seq) resolves copy number variation (CNV) at single-cell resolution, revealing tumor heterogeneity and subclonal structure. Most existing methods, however, infer only total copy number. Haplotype-resolved copy number, which captures allelic imbalance and clonal evolution, remains far less developed, largely because low coverage, allelic dropout, and technical noise in scDNA-seq make phased allelic inference substantially harder than total copy number estimation. We present CNVeil, a haplotype-aware framework that infers total, allele-specific, and chromosome-scale haplotype-resolved copy number from scDNA-seq data. CNVeil first builds robust total copy number profiles through highly variable bin selection, hierarchical clustering, subclone-aware ploidy estimation, and cross-cell consensus segmentation. Using this profile as a stable scaffold, it infers allele-specific copy number with an expectation-maximization algorithm applied to heterozygous SNP allele counts, then reconstructs haplotype-specific copy number by enforcing coherent haplotype orientation across adjacent segments via dynamic programming. We benchmarked CNVeil against 12 state-of-the-art methods, including eight total copy number callers, two allele-specific callers, and two haplotype-resolved callers, across 20 simulated and real datasets spanning six experimental settings, including high-multiplexed single-nucleus sequencing, Acoustic Cell Tagmentation (ACT), and 10x Chromium. This constitutes the largest comparative evaluation of single-cell copy number inference methods to date. CNVeil consistently outperformed existing tools in segmentation accuracy, ploidy inference, subclone identification, and allele-specific copy number estimation. In a breast cancer multi-omics (wellDR-seq) cohort, CNVeil uncovered haplotype-specific subclonal diversification invisible to total copy number analysis alone and linked allele-specific copy number states to transcriptional variation. By transforming sparse single-cell allelic signals into chromosome-scale haplotype-resolved profiles, CNVeil closes a major methodological gap and provides a scalable framework for studying tumor evolution and functional genomic heterogeneity at single-cell resolution.</jats:p>