Abstract
ABSTRACTBackgroundDiploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence. Trio binning offers an innovative solution that exploits heterozygosity for assembly. Short, parental reads are used to assign parental origin to long reads from their F1 offspring before assembly, enabling complete haplotype resolution. Trio binning could therefore provide an effective strategy for assembling highly heterozygous genomes, which are traditionally problematic, such as insect genomes. This includes the wood tiger moth (Arctia plantaginis), which is an evolutionary study system for warning colour polymorphism.FindingsWe produced a high-quality, haplotype-resolved assembly for Arctia plantaginis through trio binning. We sequenced a same-species family (F1 heterozygosity ∼1.9%) and used parental Illumina reads to bin 99.98% of offspring Pacific Biosciences reads by parental origin, before assembling each haplotype separately and scaffolding with 10X linked reads. Both assemblies are contiguous (mean scaffold N50: 8.2 Mb) and complete (mean BUSCO completeness: 97.3%), with annotations and 31 chromosomes identified through karyotyping. We used the assembly to analyse genome-wide population structure and relationships between 40 wild resequenced individuals from 5 populations across Europe, revealing the Georgian population as the most genetically differentiated with the lowest genetic diversity.ConclusionsWe present the first invertebrate genome to be assembled via trio binning. This assembly is one of the highest quality genomes available for Lepidoptera, supporting trio binning as a potent strategy for assembling heterozygous genomes. Using our assembly, we provide genomic insights into the geographic population structure of A. plantaginis.
Highlights
Diploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence
Using GenomeScope [34], we estimated the F1 offspring haploid genome size to be 590 megabase pairs (Mb) with a repeat fraction of 27% and whole-genome heterozygosity of ∼1.9% (Supplementary Fig. S3). This value was similar to our mean heterozygosity estimate of ∼1.8% in a wild, Finnish population (Supplementary Table S4; method described in Supplementary Text S2), demonstrating that our reference assembly is representative of natural variation in a wild population
By converting heterozygosity into an asset rather than a hindrance, trio binning provides an effective solution for de novo assembly of heterozygous regions, with our high-quality A. plantaginis reference genome paving the way for the use of trio binning to successfully assemble other highly heterozygous genomes
Summary
Diploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence. Trio binning could provide an effective strategy for assembling highly heterozygous genomes, which are traditionally problematic, such as insect genomes. We sequenced a same-species family (F1 heterozygosity ∼1.9%) and used parental Illumina reads to bin 99.98% of offspring Pacific Biosciences reads by parental origin, before assembling each haplotype separately and scaffolding with 10X linked reads. Most current technologies attempt to collapse parental haplotypes into a composite, haploid sequence, introducing erroneous duplications through mis-assembly of heterozygous sites as separate genomic regions. This problem is exacerbated in highly heterozygous genomes, resulting in fragmented and inflated assemblies that impede downstream analyses [3, 4]. Whilst reducing heterozygosity by inbreeding has been a frequent approach, rearing inbred lines is unfeasible and highly time consuming for many non-model systems, and resulting genomes may no longer be representative of wild populations
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