Manuka Genome Assembly using Chromosome Conformation Capture Hi-C Analysis

We have evaluated a novel strategy combining capture of chromatin interaction within the nucleus (Hi-C), next-generation sequencing and new bioinformatics methods (Proximity-Guided Assembly, PGA) for developing a near-complete pseudo-chromosome assembly of the mānuka (Lepstospermum scoparium ‘Crimson Glory’) genome. The method relies on capturing the folded conformation of chromosomes inside the nucleus and on the fact that loci that are genetically close on the same chromosome are more likely to interact in 3D space. Chromatin proximity interactions are captured through in vivo crosslinking and the resulting DNA junctions are sequenced using NGS. The PGA bioinformatics approach scaffolds the genome by arranging contigs into chromosomes in way most congruent with Hi-C data, thus maximizing the likelihood that contigs are arranged in correct order. Using PGA analysis, we inferred the location of previously unanchored contigs and created chromosome length ultra-scaffolds, enabling a ~100 times increase in the N50 scaffold length compared with assembly in the absence of this analysis. Furthermore, the Hi-C technique allowed the separation of the mānuka genome from contaminant contigs assembled from associated endophytic fungal and bacterial species. The newly assembled mānuka genome was compared to the genome of Eucalyptus grandis and high density genetic maps of mānuka constructed using genotyping-by-sequencing. The mānuka genome sequence will help shed new light on the genetic control of unique characters such as nectar and foliage biochemical composition, flowering time and disease resistance.