Abstract
At present, based on whole genome sequencing, sequences and genes annotation of the sheep (Ovis aries) Y chromosome are still absent. The isolation of Y chromosomes followed by sequencing has been approved as an effective approach to analyze this complex chromosome in other species. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts, which was successfully applied to flow-sorting chromosomes of sheep, with a focus on isolation and sequencing of the ovine Y chromosome. The isolated (~80,000) Y chromosomes were verified by fluorescence quantitative real-time polymerase chain reaction, further confirmed by fluorescence in situ hybridization, and amplified by the MALBAC method before next-generation sequencing. The sequence results indicated that 68.90% of reads were Y chromosome-related sequences as they are homologous to the bovine Y chromosome. The remaining 31.1% of reads were aligned to the sheep reference genome, including 13.57% reads to chromosome X and 6.68% to chromosome 17. Importantly, the paired-end reads that are properly aligned to the bovine Y sequence assembly accounted for 46.49%, indicating the success in the ovine Y chromosome isolation and the high quality of the Y chromosome sequences. This study not only set up a foundation for future sequencing, assembly and annotation of the ovine Y chromosome, but also provide a validated approach to overcoming difficulties in sequencing Y chromosome in other mammalian species.
Highlights
Flow cytometric sorting of chromosomes is considered a powerful tool for chromosome research due to its ability to isolate particular chromosomes with good purity and quantities
This study demonstrates the potential application of flow cytometry for genome and chromosome research and lays a foundation for future sequencing and annotation of the ovine Y chromosome
Epithelial cells around the explants were first seen at 48 h in the primary skin tissue culture, and primary skin fibroblasts were observed on the outer layer of epithelial cells on day 5 (Fig. 1a)
Summary
Flow cytometric sorting of chromosomes is considered a powerful tool for chromosome research due to its ability to isolate particular chromosomes with good purity and quantities. In recent years, sequencing technology has advanced to permit the sequencing of single chromosomes of interest isolated by flow cytometric sorting. This method has been successfully applied in humans[5,6], gorillas[1], Chinese hamsters[7], and some plant species[8,9,10,11]. We isolated and enriched sheep Y chromosomes by flow cytometric sorting and performed NGS of sorted Y chromosomes, which laid a foundation for the sequencing, assembly, and analysis of the sheep Y chromosome. We successfully isolated and enriched ovine Y chromosomes by flow cytometric sorting and performed NGS. This study demonstrates the potential application of flow cytometry for genome and chromosome research and lays a foundation for future sequencing and annotation of the ovine Y chromosome
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