HapAsmbl: A reference‐aided pipeline for assembling haplotypes in Nanopore amplicon sequence data of polymorphic populations

Abstract Premise Advances in long‐read sequencing offer new possibilities to investigate haplotype diversity across multiple genes in plants and other taxa through multi‐locus, long‐read amplicon sequencing (multi‐locus LRAS). Despite this progress, there is a notable absence of dedicated bioinformatics pipelines for assembling diploid haplotypes of heterozygous individuals from such multi‐locus LRAS datasets, which is required for highly polymorphic populations. Methods We first evaluated various de novo and reference‐based assembly methods, culminating in a custom pipeline (HapAsmbl) to assemble haplotypes from Oxford Nanopore Technologies (ONT) LRAS data of five flowering genes ( FT3 , FTL9 , VRN1 , VRN2A , and VRN2B ) generated from perennial ryegrass, a highly heterozygous species. After verifying the efficacy using a simulated heterozygous dataset, the HapAsmbl pipeline was used to explore haplotype diversity of CO , FT3 , and VRN1 across multiple ryegrass populations. Results HapAsmbl outperformed existing tools by reliably reconstructing diploid haplotypes across multiple loci, enabling efficient haplotype characterization and novel allele discovery in genetically diverse populations. Discussion HapAsmbl simplifies haplotype resolution from complex LRAS datasets from heterozygous individuals, allowing routine use of ONT long‐read sequencing for scalable haplotype analysis. HapAsmbl will enable researchers to uncover novel alleles and relate these to phenotype, supporting plant‐breeding efforts in non‐model crops.