Chromosome-level and haplotype-resolved genome assembly enabled by high-throughput single-cell sequencing of gamete genomes

Generating haplotype-resolved, chromosome-level assemblies of heterozygous genomes remains challenging. To address this, we developed gamete binning, a method based on single-cell sequencing of hundreds of haploid gamete genomes, which enables the separation of conventional long sequencing reads into two haplotype-specific read sets. After independently assembling the reads of each haplotype, the contigs are scaffolded to chromosome-level using a genetic map derived from the recombination patterns within the same gamete genomes. As a proof-of-concept, we assembled the two genomes of a diploid apricot tree supported by the analysis of 445 pollen genomes. Both assemblies (N50: 25.5 and 25.8 Mb) featured a haplotyping precision of >99% and were accurately scaffolded to chromosome-level as reflected by high levels of synteny to closely-related species. These two assemblies allowed for first insights into haplotype diversity of apricot and enabled the identification of non-allelic crossover events introducing severe chromosomal anomalies in 1.6% of the pollen genomes.