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

Area of Science:

• Genomics and Bioinformatics
• Plant Genetics
• Population Genetics

Background:

• Long-read sequencing technologies, such as Oxford Nanopore Technologies (ONT), have advanced multi-locus, long-read amplicon sequencing (LRAS) for studying haplotype diversity.
• A gap exists in bioinformatics pipelines for assembling diploid haplotypes from multi-locus LRAS data, particularly for heterozygous and highly polymorphic plant populations.

Purpose of the Study:

• To develop and evaluate a bioinformatics pipeline for assembling diploid haplotypes from multi-locus LRAS data.
• To enable scalable haplotype analysis and novel allele discovery in heterozygous plant individuals.

Main Methods:

• Evaluated various de novo and reference-based assembly methods.
• Developed a custom pipeline, HapAsmbl, to assemble haplotypes from ONT LRAS data of flowering genes in perennial ryegrass.
• Validated HapAsmbl using simulated heterozygous datasets and applied it to analyze haplotype diversity in multiple ryegrass populations.

Main Results:

• HapAsmbl demonstrated superior performance in reliably reconstructing diploid haplotypes across multiple loci compared to existing tools.
• The pipeline enabled efficient haplotype characterization and the discovery of novel alleles in genetically diverse populations.
• Successfully applied HapAsmbl to explore haplotype diversity of key flowering genes in perennial ryegrass.

Conclusions:

• HapAsmbl simplifies haplotype resolution from complex LRAS datasets, facilitating routine use of ONT long-read sequencing for haplotype analysis.
• The pipeline supports researchers in uncovering novel alleles and linking them to plant phenotypes, aiding plant breeding efforts, especially in non-model crops.