Mitigating assembly and switch errors in phased genomes of polar fishes reveals haplotype diversity in copy number of antifreeze protein genes

Abstract

Phased genomes and pangenomes are enhancing our understanding of genetic variation. Accurate phasing and assembly in repetitive regions of the genome remain challenging, however. Addressing this obstacle is crucial for studying structural genomic variation such as copy number variations (CNVs) common to repetitive regions. Polar fishes, for example, evolved repetitive tandem arrays of antifreeze protein (AFP) genes that facilitated adaptation to freezing and expanded in copy number in colder environments. AFP CNVs remain poorly characterized in polar fishes and may be illuminated by haplotype-aware approaches. We performed long-read sequencing of two polar fishes in the suborder Zoarcoidei and leveraged published long-read data to assemble phased genomes. We developed a workflow to measure haplotype diversity in CNV while controlling for misassembly and switch errors--a change from one parental haplotype to another in a contiguous assembly. We present gfa_parser, which computes and extracts all possible contiguous sequences for phased or primary assemblies from graphical fragment assembly files, and switch_error_screen, which flags potential switch errors. gfa_parser revealed that assembly uncertainty was ubiquitous across AFP array haplotypes and that standard processing of graphical fragment assemblies can bias measurement of haplotype CNVs. We detected no switch errors in AFP arrays. After controlling for misassembly and switch error, we detected haplotype diversity of AFP CNVs in all studied polar Zoarcoidei species and in 60% of AFP arrays. Intraindividual haplotype diversity spanned differences of 3-16 copies. Our workflow revealed intraspecific genomic diversity in zoarcoids that likely fueled evolution of AFP copy number across temperature.