While the cost and time for assembling a genome has drastically decreased, it still remains a challenge to assemble a highly contiguous genome. These challenges are rapidly being overcome by the integration of long-read sequencing technologies. Here, we use long-read sequencing to improve the contiguity of the threespine stickleback fish (Gasterosteus aculeatus) genome, a prominent genetic model species. Using Pacific Biosciences sequencing, we assembled a highly contiguous genome of a freshwater fish from Paxton Lake. Using contigs from this genome, we were able to fill over 76% of the gaps in the existing reference genome assembly, improving contiguity over five-fold. Our gap filling approach was highly accurate, validated by 10X Genomics long-distance linked-reads. In addition to closing a majority of gaps, we were able to assemble segments of telomeres and centromeres throughout the genome. This highlights the power of using long sequencing reads to assemble highly repetitive and difficult to assemble regions of genomes. This latest genome build has been released through a newly designed community genome browser that aims to consolidate the growing number of genomics datasets available for the threespine stickleback fish.

AbstractWhile the cost and time for assembling a genome have drastically reduced, it still remains a challenge to assemble a highly contiguous genome. These challenges are rapidly being overcome by the integration of long-read sequencing technologies. Here, we use long sequencing reads to improve the contiguity of the threespine stickleback fish (Gasterosteus aculeatus) genome, a prominent genetic model species. Using Pacific Biosciences sequencing, we were able to fill over 76% of the gaps in the genome, improving contiguity over five-fold. Our approach was highly accurate, validated by 10X Genomics long-distance linked-reads. In addition to closing a majority of gaps, we were able to assemble segments of telomeres and centromeres throughout the genome. This highlights the power of using long sequencing reads to assemble highly repetitive and difficult to assemble regions of genomes. This latest genome build has been released through a newly designed community genome browser that aims to consolidate the growing number of genomics datasets available for the threespine stickleback fish.

Objective: To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. Design: Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. Setting: Multiple academic laboratory settings. Patient(s): Not applicable. Intervention(s): Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measure(s): Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. Result(s): Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. Conclusion(s):This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.

Double strand break repair during meiosis is normally achieved using the homologous chromosome as a repair template. Heteromorphic sex chromosomes have reduced sequence homology with one another, presenting unique challenges to the repair of double strand breaks. Our understanding of how heteromorphic sex chromosomes behave during meiosis has been largely limited to the ancient sex chromosomes of mammals, where the X and Y differ markedly in overall structure and gene content. Consequently, pairing of the X and Y chromosomes is limited to a small pseudoautosomal region. It remains unclear how more recently evolved sex chromosomes, that share considerably more sequence homology with one another, pair and repair double strand breaks during meiosis. One possibility is barriers to pairing evolve rapidly. Alternatively, recently evolved sex chromosomes may exhibit pairing and double strand break repair that more closely resembles that of their autosomal ancestors. Here we use the recently evolved X and Y chromosomes of the threespine stickleback fish (Gasterosteus aculeatus) to study patterns of pairing and double stranded break formation using molecular cytogenetics. We found that the sex chromosomes of threespine stickleback fish do not pair exclusively in the pseudoautosomal region. Instead, the chromosomes fully pair in a non-homologous fashion. To achieve this, the X chromosome underwent synaptic adjustment during pachytene to match the axis length of the Y chromosome. Double strand break formation and repair rate also matched that of the autosomes. Our results highlight that recently evolved sex chromosomes exhibit meiotic behavior that is reminiscent of autosomes and argues for further work to identify the homologous templates that are used to repair double strand breaks on the X and Y chromosomes.

Double strand break repair during meiosis is normally achieved using the homologous chromosome as a repair template. Heteromorphic sex chromosomes have reduced sequence homology with one another, presenting unique challenges to the repair of double strand breaks.Our understanding of how heteromorphic sex chromosomes behave during meiosis has been largely limited to the ancient sex chromosomes of mammals, where the X and Y differ markedly in overall structure and gene content. Consequently, pairing of the X and Y chromosomes is limited to a small pseudoautosomal region. It remains unclear how more recently evolved sex chromosomes, that share considerably more sequence homology with one another, pair and repair double strand breaks during meiosis. One possibility is barriers to pairing evolve rapidly.Alternatively, recently evolved sex chromosomes may exhibit pairing and double strand break repair that more closely resembles that of their autosomal ancestors. Here we use the recently evolved X and Y chromosomes of the threespine stickleback fish (Gasterosteus aculeatus) to study patterns of pairing and double stranded break formation using molecular cytogenetics. We found that the sex chromosomes of threespine stickleback fish do not pair exclusively in the pseudoautosomal region. Instead, the chromosomes fully pair in a non-homologous fashion. To achieve this, the X chromosome underwent synaptic adjustment during pachytene to match the axis length of the Y chromosome. Double strand break formation and repair rate also matched that of the autosomes. Our results highlight that recently evolved sex chromosomes exhibit meiotic behavior that is reminiscent of autosomes and argues for further work to identify the homologous templates that are used to repair double strand breaks on the X and Y chromosomes.