Methylation-aware long-read phasing significantly improves genome-wide haplotype reconstruction

Abstract

Haplotypes are linear sequences of co-inherited alleles along individual chromosomes and are central to genetic mapping, clinical variant interpretation, and inference of population history. However, accurate genome-wide haplotype reconstruction remains challenging. Long-read sequencing has the potential to dramatically improve haplotype inference, but existing methods do not directly leverage all the information embedded in these data. Here, we present LongHap, a read-based phasing method that integrates sequence and 5-methylcytosine (5mC) information in a unified probabilistic framework. By leveraging differentially methylated sites, LongHap resolves phase relationships between variants that are inaccessible to sequence-based approaches alone. Across multiple datasets and sequencing platforms, LongHap increases phase block lengths by up to 30% while substantially reducing switch error rates. LongHap rigorously embeds complex structural variants into the broader haplotype context using loopy belief propagation, enabling improved phasing of INDELs and other variant classes that are inherently difficult to resolve. Methylation-aware phasing also improves the accuracy and contiguity of haplotypes spanning rare variants and structurally complex, medically relevant genes across diverse ancestries, facilitating the interpretation of compound heterozygosity and haplotype-specific regulatory architectures. These results establish methylation-aware phasing as a general framework for improving genome-wide haplotype reconstruction, with broad applications across genetics and genomics.