A complete pipeline enables haplotyping and phasing macrohaplotype in long sequencing reads for polyploidy samples and a multi-source DNA mixture

• NCJ Number: 308426
• Journal: Electrophoresis Volume: Online Dated: January 2024
• Author(s): Xuewen Wang; Melissa Muenzler; Jonathan King; Muyi Liu; Hongmin Li; Bruce Budowle; Jianye Ge
• Date Published: January 2024
• Grant Number(s): 15PNIJ-21-GG-04159-RESS
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Enhanced mixture interpretation with long-read DNA sequencing

Annotation

This paper reports on a research study that resulted in the development of a bioinformatics software in which the targeted loci were genotyped and combined with novel algorithms to call macrohaplotypes from long reads.

Abstract

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, the authors 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 approximately eight-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. (Published Abstract Provided)