Yoshitake Kazutoshi, Ishikawa Asano, Yonezawa Ryo, Kinoshita Shigeharu, Kitano Jun, Asakawa Shuichi
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, Japan.
Ecological Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
NAR Genom Bioinform. 2022 Mar 31;4(2):lqac026. doi: 10.1093/nargab/lqac026. eCollection 2022 Jun.
It is still difficult to construct the genomes of higher organisms as their genome sequences must be extended to the length of the chromosome by linkage analysis. In this study, we attempted to provide an innovative alternative to conventional linkage analysis by devising a method to genotype sperm using 10× Genomics single-cell genome sequencing libraries to generate a linkage map without interbreeding individuals. A genome was assembled using sperm from the Japanese stickleback , with single-cell genotyping yielding 1 864 430 very dense hetero-SNPs and an average coverage per sperm cell of 0.13×. In total, 1665 sperm were used, which is an order of magnitude higher than the number of recombinations used for conventional linkage analysis. We then improved the linkage analysis tool scaffold extender with low depth linkage analysis (SELDLA) to analyze the data according to the characteristics of the single-cell genotyping data. Finally, we were able to determine the chromosomal location (97.1%) and orientation (64.4%) of the contigs in the 456 Mb genome of , sequenced using nanopores. This method promises to be a useful tool for determining the genomes of non-model organisms for which breeding systems have not yet been established by linkage analysis.
构建高等生物的基因组仍然很困难，因为它们的基因组序列必须通过连锁分析扩展到染色体的长度。在本研究中，我们试图通过设计一种使用10×基因组学单细胞基因组测序文库对精子进行基因分型的方法，来提供一种替代传统连锁分析的创新方法，从而在不进行个体杂交的情况下生成连锁图谱。我们使用日本刺鱼的精子组装了一个基因组，单细胞基因分型产生了1864430个非常密集的杂合单核苷酸多态性（hetero-SNPs），每个精子细胞的平均覆盖率为0.13×。总共使用了1665个精子，这比传统连锁分析中使用的重组数量高出一个数量级。然后，我们改进了连锁分析工具低深度连锁分析支架扩展器（SELDLA），以根据单细胞基因分型数据的特征分析数据。最后，我们能够确定使用纳米孔测序的456 Mb基因组中重叠群的染色体位置（97.1%）和方向（64.4%）。这种方法有望成为一种有用的工具，用于确定那些尚未通过连锁分析建立育种系统的非模式生物的基因组。
