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HapAsmbl: A reference-aided pipeline for assembling haplotypes in Nanopore amplicon sequence data of polymorphic
Ayodele Oluwaseyi Fakoya1, Augustine Chen1, Rowan Paul Herridge1
1Department of Biochemistry University of Otago Dunedin New Zealand.
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.
