Chromosome-level genome construction of a Japanese stickleback species using ultra-dense linkage analysis using single-cell sequencing of sperms

Abstract

The presence of high quality genomes at the chromosome level is very useful in t he search for the causal genes of mutants and in genetic breeding. The advent of n ext-generation sequencers has made it easier to decode genomes, but it is still di fficult to construct the genomes of higher organisms. In order to construct the ge nome of a higher organism, the genome sequence of the organism is extended to the length of the chromosome by linkage analysis after assembly and scaffolding. Howev er, in the past linkage analysis, it was difficult to make a high-density linkage map, and it was not possible to analyze organisms without an established breeding system. As an innovative alternative to conventional linkage analysis, we devised a method for genotyping sperm using 10x single-cell genome (CNV) sequencing librar ies to generate a linkage map without interbreeding individuals. The genome was co nstructed using sperm from Gasterosteus nipponicus, and single-cell genotyping yie lded 1,864,430 very dense hetero-SNPs. The average coverage per sperm cell is 0.13 x. The number of sperm used is 1,738, which is an order of magnitude higher than t he number of sperm used for conventional linkage analysis. We have improved the li nkage analysis tool SELDLA (Scaffold Extender with Low Depth Linkage Analysis) so that we can analyze the data in accordance with the characteristics of single-cell genotyping data. Finally, we were able to determine the location and orientation o n the chromosome for 85.6% of the contigs in the 456 Mbase genome of Gasterosteus nipponicus sequenced in nanopores. A total of 95.6% of the contigs in which a cros s-reaction was detected within the contigs.