Abuelanin, M.; Kaya, G.; Lake, J. A.; Lambert, C.; Wu, M. V.; Berendzen, K.; Krasheninnikova, K.; Wood, J. M.; Solomon, N. G.; Donaldson, Z. R.; Bales, K. L.; Howe, K.; Korlach, J.; Manoli, D. S.; Tollkuhn, J.; Dennis, M. Y.

High-quality reference genomes are essential to effectively characterize genomic drivers of speciation, phenotypic diversity, and disease causality. Larger complex genomes often require integration of long-read DNA sequencing with additional genomic data, such as chromosome conformation capture (Hi-C or CiFi) to generate phased chromosome-scale assemblies, however this requires multiple sequencing platforms (in the case of Hi-C) or the construction of multiple long-read sequencing libraries. Here, we devise a strategy that combines PacBio HiFi and CiFi sequencing in a single library and run to efficiently produce high-quality contiguous chromosome-scale diploid genome assemblies. We apply this approach to liver tissue from single individuals of prairie vole (Microtus ochrogaster) and meadow vole (Microtus pennsylvanicus), generating haplotype-resolved, chromosome-scale 2.3 Gbp genomes with QV~62, and 99.3% BUSCO completeness. Comparing the two new genomes identifies complex structural changes impacting Avpr1a, previously implicated in pair bonding, including a species-specific duplication missing from the existing prairie vole reference genome. These divergent genomic features offer new avenues of investigation related to behavioral divergence between prairie and meadow voles. This single-library approach facilitates a simplified and more affordable assembly workflow, producing near-complete genomes of diverse species using one sequencing platform.