A complete pipeline enables haplotyping and phasing macrohaplotype in long sequencing reads for polyploidy samples...

Macrohaplotype combines multiple types of phased DNA variants, increasing forensic discrimination power. High-quality long-sequencing reads, for example, PacBio HiFi reads, provide data to detect macrohaplotypes in multiploidy and DNA mixtures. However, the bioinformatics tools for detecting macrohaplotypes are lacking. In this study, we developed a bioinformatics software, MacroHapCaller, in which targeted loci (i.e., short TRs [STRs], single nucleotide polymorphisms, and insertion and deletions) are genotyped and combined with novel algorithms to call macrohaplotypes from long reads. MacroHapCaller uses physical phasing (i.e., read-backed phasing) to identify macrohaplotypes, and thus it can detect multi-allelic macrohaplotypes for a given sample. MacroHapCaller was validated with data generated from our designed targeted PacBio HiFi sequencing pipeline, which sequenced ∼8-kb amplicon regions harboring 20 core forensic STR loci in human benchmark samples HG002 and HG003. MacroHapCaller also was validated in whole-genome long-read sequencing data. Robust and accurate genotyping and phased macrohaplotypes were obtained with MacroHapCaller compared with the known ground truth. MacroHapCaller achieved a higher or consistent genotyping accuracy and faster speed than existing tools HipSTR and DeepVar. MacroHapCaller enables efficient macrohaplotype analysis from high-throughput sequencing data and supports applications using discriminating macrohaplotypes.