Physical separation of haplotypes in dikaryons allows benchmarking of phasing accuracy in Nanopore and HiFi assemblies with Hi-C data

Abstract

Background Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. Results We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30–40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. Conclusions This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

A phased chromosome-level genome and full mitochondrial sequence for the dikaryotic myrtle rust pathogen, Austropuccinia psidii

“…Thirteen hap1 and twelve hap2 scaffolds are telomere to telomere, indicating likely full-length chromosomes (Table 1 and Figure 2). Comparison to the wheat leaf rust (strain Pt76) genome (Duan et al . 2022) shows the same haploid chromosome count of n =18, with many of the chromosomes presenting a dominant syntenic relationship with a single myrtle rust scaffold, despite the significant enlargement in chromosome size (Figure 2).…”

“…Genes on the forward strand are marked above the centre-line, with reverse strand genes below. (A) Pairs of phased haploid genomes for A. psidii v2 and wheat leaf rust chromosomes, Pt76_A and B (Duan et al . 2022).…”

“…2021). However we present a much improved assembly with high synteny of conserved genes between the haploid chromosomes, and compared to the genome for wheat leaf rust, Puccinia triticina (Pt76) (Duan et al . 2022) (Figure 2).…”

“…These findings have been recently further confirmed with new technology that has enabled phasing and scaffolding of genomes for wheat leaf rust (Wu et al . 2021; Duan et al . 2022) and stripe rust (Schwessinger et al .…”

“…Code for establishing and visualising chromosome synteny based on shared BUSCO genes has been publicly released as a new tool, ChromSyn, under a GNU General Public License v3.0 and is available on GitHub (https://github.com/slimsuite/chromsyn) . Synteny of the two A. psidii haploid genomes was analysed with ChromSyn v0.9.3 and further comparisons were also made against the recently published fully phased, chromosome-level wheat leaf rust genome, Puccinia triticina (Pt76) (Duan et al . 2022).…”