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Universal dynamics of cohesin-mediated loop extrusion 聚合酶介导的环挤压的通用动力学
Pub Date : 2024-08-10 DOI: 10.1101/2024.08.09.605990
Thomas Sabate, Benoit Lelandais, Marie-Cecile Robert, Michael Szalay, Jean-Yves Tinevez, Edouard Bertrand, Christophe Zimmer
Most animal genomes are partitioned into Topologically Associating Domains (TADs), created by cohesin-mediated loop extrusion and defined by convergently oriented CTCF sites. The dynamics of loop extrusion and its regulation remains poorly characterized in vivo. Here, we tracked TAD anchors in living human cells to visualize and quantify cohesin-dependent loop extrusion across multiple endogenous genomic regions. We show that TADs are dynamic structures whose anchors are brought in proximity about once per hour and for 6-19 min (~16% of the time). TADs are continuously subjected to extrusion by multiple cohesin complexes, extruding loops at ~0.1 kb/s. Remarkably, despite strong differences of Hi-C patterns between the chromatin regions, their dynamics is consistent with the same density, residence time and speed of cohesin. Our results suggest that TAD dynamics is governed primarily by CTCF site location and affinity, which allows genome-wide predictive models of cohesin-dependent interactions.
大多数动物基因组都被划分为拓扑关联区(TAD),由凝聚素介导的环挤压形成,并由趋同取向的 CTCF 位点定义。在体内,环挤压及其调控的动态特征仍然不甚明了。在这里,我们追踪了活人细胞中的 TAD 锚点,以可视化和量化多个内源基因组区域中依赖于粘合素的环挤出。我们发现,TADs 是一种动态结构,其锚大约每小时靠近一次,持续时间为 6-19 分钟(约占 16%)。TADs 不断受到多个凝聚素复合物的挤压,以每秒约 0.1 kb 的速度挤出环。值得注意的是,尽管染色质区域之间的 Hi-C 模式差异很大,但它们的动态与相同的密度、停留时间和凝聚素速度是一致的。我们的研究结果表明,TAD 的动态主要受 CTCF 位点位置和亲和力的支配,这使得全基因组范围内依赖于凝聚素的相互作用的预测模型成为可能。
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引用次数: 0
Non-destructive DNA extraction from specimens and environmental samples using DESS preservation solution for DNA barcoding 使用 DESS 保存解决方案从标本和环境样本中无损提取 DNA,用于 DNA 条形码分析
Pub Date : 2024-08-10 DOI: 10.1101/2024.08.09.607403
Eri Ogiso-Tanaka, Minako Abe Ito, Daisuke Shimada
DESS is a widely used storage solution for the preservation of DNA from biological tissue samples. DESS comprises 20% dimethyl sulfoxide, 250 mM ethylenediaminetetraacetic acid, and saturated sodium chloride, and its efficacy has been confirmed in various taxa and tissues. DESS enables the stable, long-term preservation of both sample morphology and DNA. However, to access the DNA, excising a portion of the sample was necessary. Although DNA is a valuable source of information for species identification, DNA extraction can result in the loss of an entire sample or segment, especially in small-sized organisms, thereby compromising specimen value. Therefore, establishing non-destructive DNA extraction techniques is imperative. Thus, this paper presents a protocol for conducting non-destructive DNA extraction and DNA barcoding using a portion of the DESS supernatant obtained from a nematode specimen. This method was successfully employed for DNA barcoding of nematodes that were stored in DESS at room temperature (-10~35˚C) for ten years. Moreover, the method can be potentially applied in the preservation and non-destructive extraction of DNA from specimens of various species. Following sample collection, a bulk environmental sample from sediment and seagrass is immediately immersed in DESS in the field. Subsequently, DNA is extracted from the supernatant solution, allowing non-destructive DNA barcoding. Overall, this paper presents comprehensive protocols for DNA extraction from DESS supernatants and demonstrates their practical application using meiofauna (small animals) and diatoms as examples.
DESS 是一种广泛使用的生物组织样本 DNA 保存液。DESS 由 20% 二甲基亚砜、250 mM 乙二胺四乙酸和饱和氯化钠组成,其功效已在不同类群和组织中得到证实。DESS 可以长期稳定地保存样本形态和 DNA。不过,要获取 DNA,必须切除部分样本。虽然 DNA 是物种鉴定的宝贵信息来源,但 DNA 提取可能会导致整个样本或样本片段的损失,尤其是在小型生物体中,从而影响标本的价值。因此,建立非破坏性的 DNA 提取技术势在必行。因此,本文介绍了一种利用从线虫标本中获得的部分 DESS 上清液进行非破坏性 DNA 提取和 DNA 条形码的方法。该方法被成功地用于对在室温(-10~35˚C)下储存在 DESS 中的线虫进行 DNA 条形编码。此外,该方法还可用于保存和无损提取不同物种标本中的 DNA。样本采集完成后,在野外立即将沉积物和海草的大量环境样本浸入 DESS 中。随后,从上清液中提取 DNA,从而实现非破坏性 DNA 条形编码。总之,本文介绍了从 DESS 上清液中提取 DNA 的综合方案,并以小型动物和硅藻为例展示了其实际应用。
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引用次数: 0
Optimising future rhino population management strategies using insights from genetic health assessments across India 利用对印度各地遗传健康评估的深入了解,优化未来犀牛种群管理战略
Pub Date : 2024-08-09 DOI: 10.1101/2024.08.09.607316
Tista Ghosh, Parikshit Kakati, Amit Sharma, Samrat Mondol
Various species conservation paradigms are facing enormous challenges during the ongoing Anthropocene. While the widely-used reintroduction/translocation-based approaches have supported many endangered species population recoveries, they seldom use detailed genetic information during initial planning. The Indian greater one-horned rhino typifies such assisted migration-driven species recovery, but currently facing long-term survival concerns due to their mostly small, isolated populations reaching respective carrying capacities. We assessed nation-wide rhino genetic health, identified suitable source populations and provided future translocation scenarios for all extant and proposed rhino habitats. Analyses with 504 unique rhino genotypes across all seven Indian rhino-bearing parks revealed six genetically-isolated populations with overall moderately low genetic diversity. Our results showed that Kaziranga and Manas NPs (Assam) to have the best rhino genetic health, whereas Jaldapara and Gorumara NPs (West Bengal) undergoing strong genetic erosions. Forward genetic simulations suggested that annual supplementation efforts from only few Assam rhino populations (Kaziranga NP, Orang NP and Pobitora WLS) are best suited for genetic rescue of most of the extant populations. Overall, the genetic diversity and differentiation patterns mimics the complex evolutionary history and individual recovery histories. We suggest park-specific management solutions (ranging from protection measures, grassland restoration, livestock and conflict management, regular supplementation events etc.) to ensure the species long-term persistence and prevent the alarming loss of grassland habitats and its associated biodiversity. We insist on utilising such genetic health indices-driven population management solutions to identify targeted mitigative measures in other species.
在当前的人类世,各种物种保护模式都面临着巨大的挑战。虽然广泛使用的基于重引/迁移的方法支持了许多濒危物种的种群恢复,但在最初的规划中却很少使用详细的遗传信息。印度大独角犀牛就是这种辅助迁移驱动的物种恢复的典型代表,但由于其种群大多较小且孤立,目前已达到各自的承载能力,因此面临着长期生存问题。我们对全国范围内的犀牛基因健康状况进行了评估,确定了合适的源种群,并为所有现存和拟建的犀牛栖息地提供了未来的迁移方案。通过对印度所有 7 个犀牛栖息地的 504 个独特犀牛基因型进行分析,我们发现了 6 个基因隔离的种群,其遗传多样性总体上处于中等偏低的水平。我们的研究结果表明,卡齐兰加和马纳斯国家公园(阿萨姆邦)的犀牛基因健康状况最好,而贾尔达帕拉和戈鲁马拉国家公园(西孟加拉邦)的犀牛基因正在遭受严重侵蚀。前瞻性遗传模拟表明,只有少数阿萨姆邦犀牛种群(卡齐兰加保护区、奥兰加保护区和波比托拉野生动物保护区)的年度补充工作最适合拯救大多数现存种群的遗传。总体而言,遗传多样性和分化模式模拟了复杂的进化史和个体恢复史。我们提出了针对公园的管理解决方案(包括保护措施、草地恢复、牲畜和冲突管理、定期补充活动等),以确保该物种的长期存在,防止草地栖息地及其相关生物多样性的惊人丧失。我们坚持利用这种遗传健康指数驱动的种群管理解决方案,为其他物种确定有针对性的缓解措施。
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引用次数: 0
Whole genome sequence improvement with pedigree information and reference genotypic profiles, demonstrated in outcrossing apple 利用血统信息和参考基因型图谱进行全基因组序列改良,在苹果外交中得到验证
Pub Date : 2024-08-09 DOI: 10.1101/2024.08.08.607141
Stijn Vanderzande, Cameron Peace, Eric van de Weg
Understanding the quality of a whole genome sequence (WGS) is important for its further use. Most WGS quality evaluations are based on bioinformatic quality metrics such as the N50 score, BUSCO score, and number of contigs and scaffolds present, yet genetic information considering principles of inheritance could be used to evaluate and improve assembly and phasing. Furthermore, WGS and genome resequencing data of related individuals could provide useful information when large chromosomal segments are shared with the target individual through common ancestry. Here, we show how high-quality, phased, genome-wide genotypic information is useful to evaluate the quality of a WGS. We provide an R-tool to routinely conduct such quality evaluations. The script also provides a method to accurately determine the WGS positions of reference SNP markers, which is needed for integration of SNP array-based genotypic data sets with WGS data, and the identification and comparison of segments across WGSs that are shared by descent. Finally, we provide suggestions on how such sharing can be used to evaluate and improve new WGSs. The approach is demonstrated in apple, for which improvements in WGS quality are evident from the first collapsed WGS with many inconsistencies in genetic marker order and genotype scores, through well-assembled haploid WGSs, to incorrectly and correctly phased diploid WGSs. This study shows that homozygous regions might need extra attention in phased WGSs and that further improvements to phased WGSs can be achieved by grouping chromosomes of single parental origin into the same haplome.
了解全基因组序列(WGS)的质量对其进一步使用非常重要。大多数 WGS 质量评估都是基于生物信息质量指标,如 N50 分数、BUSCO 分数以及等位基因和支架的数量,但考虑到遗传原理的遗传信息也可用于评估和改进组装和分期。此外,当目标个体通过共同祖先共享大的染色体片段时,相关个体的 WGS 和基因组重测序数据也能提供有用的信息。在这里,我们展示了高质量、分期的全基因组基因型信息如何有助于评估 WGS 的质量。我们提供了一个 R 工具,用于常规进行此类质量评估。该脚本还提供了一种准确确定参考 SNP 标记的 WGS 位置的方法,这对于基于 SNP 阵列的基因型数据集与 WGS 数据的整合,以及识别和比较各 WGS 之间共享的世系片段都是必需的。最后,我们就如何利用这种共享来评估和改进新的 WGS 提出了建议。我们在苹果中演示了这种方法,从遗传标记顺序和基因型得分存在许多不一致之处的第一个折叠 WGS,到组装良好的单倍体 WGS,再到分期错误和分期正确的二倍体 WGS,WGS 质量的提高是显而易见的。这项研究表明,在分阶段 WGS 中可能需要格外注意同源区,将单亲来源的染色体归入同一单倍体可进一步改进分阶段 WGS。
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引用次数: 0
Drosophila ring chromosomes interact with sisters and homologs to produce anaphase bridges in mitosis. 果蝇环状染色体与姐妹染色单体和同源染色体相互作用,在有丝分裂过程中产生无丝分裂桥。
Pub Date : 2024-08-09 DOI: 10.1101/2024.08.08.607186
Ho-Chen Lin, Mary M Golic, Hunter J Hill, Katherine F Lemons, Truc T Vuong, Madison Smith, Forrest T Golic, Kent G Golic
Ring chromosomes are known in many eukaryotic organisms, including humans. They are typically associated with a variety of maladies, including abnormal development and lethality. Underlying these phenotypes are anaphase chromatin bridges that can lead to chromosome loss, nondisjunction and breakage. By cytological examination of ring chromosomes in Drosophila melanogaster we identified five causes for anaphase bridges produced by ring chromosomes. Catenation of sister chromatids is the most common cause and these bridges frequently resolve during anaphase, presumably by the action of topoisomerase II. Sister chromatid exchange and chromosome breakage followed by sister chromatid union also produce anaphase bridges. Mitotic recombination with the homolog was rare, but was another route to generation of anaphase bridges. Most surprising, was the discovery of homolog capture, where the ring chromosome was connected to its linear homolog in anaphase. We hypothesize that this is a remnant of mitotic pairing and that the linear chromosome is connected to the ring by multiple wraps produced through the action of topoisomerase II during establishment of homolog pairing. In support, we showed that in a ring/ring homozygote the two rings are frequently catenated in mitotic metaphase, a configuration that requires breaking and rejoining of at least one chromosome.
包括人类在内的许多真核生物都存在环状染色体。环状染色体通常与多种疾病相关,包括发育异常和致死。这些表型的基础是可导致染色体缺失、非连接和断裂的无丝分裂染色质桥。通过对黑腹果蝇的环状染色体进行细胞学检查,我们确定了环状染色体产生无丝期桥的五种原因。姐妹染色单体的卡合是最常见的原因,这些桥经常在无丝分裂过程中消失,可能是在拓扑异构酶 II 的作用下消失的。姐妹染色单体交换和染色体断裂后姐妹染色单体结合也会产生无丝分裂桥。同源染色体的有丝分裂重组很少见,但这是产生无丝分裂桥的另一个途径。最令人惊讶的是同源物捕获的发现,即环状染色体在无丝分裂期与其线性同源物相连。我们推测,这是有丝分裂配对的残余,在同源染色体配对建立过程中,通过拓扑异构酶 II 的作用,线性染色体与环状染色体通过多重缠绕连接在一起。作为佐证,我们发现在环/环同源基因中,两个环经常在有丝分裂分裂相中结合,这种构型需要至少一条染色体的断裂和重合。
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引用次数: 0
A newly evolved gene is essential for efficient sperm entry into eggs in Drosophila melanogaster 一个新进化的基因对黑腹果蝇精子有效进入卵子至关重要
Pub Date : 2024-08-09 DOI: 10.1101/2024.08.08.607187
Sara Y Guay, Prajal H Patel, Jonathon M Thomalla, Kerry L McDermott, Jillian M O'Toole, Sarah E Arnold, Sarah J Obrycki, Mariana F Wolfner, Geoffrey D Findlay
New genes arise through a variety of evolutionary processes and provide raw material for adaptation in the face of both natural and sexual selection. De novo evolved genes emerge from previously non-protein-coding DNA sequences, and many such genes are expressed in male reproductive structures. In Drosophila melanogaster, several putative de novo genes have evolved essential roles in spermatogenesis, but whether such genes can also impact sperm function beyond the male has not been investigated. We identified a putative de novo gene, katherine johnson (kj), that is required for high levels of male fertility. Males that do not express kj produce and transfer sperm that are stored normally in females, but sperm from these males enter eggs with severely reduced efficiency. Using a tagged transgenic rescue construct, we observed that KJ protein localizes to the nuclear periphery in various stages of spermatogenesis, but is not detectable in mature sperm. These data suggest that kj exerts an effect on sperm development, the loss of which results in reduced fertilization ability. While previous bioinformatic analyses suggested the kj gene was restricted to the melanogaster group of Drosophila, we identified putative orthologs with conserved synteny, male-biased expression, and predicted protein features across the genus, as well as instances of gene loss in some lineages. Thus, kj potentially arose in the Drosophila common ancestor and subsequently evolved an essential role in D. melanogaster. Our results demonstrate a new aspect of male reproduction that has been shaped by new gene evolution and provide a molecular foothold for further investigating the mechanism of sperm entry into eggs in Drosophila.
新基因通过各种进化过程产生,并在自然选择和性选择的作用下为适应性提供原材料。新进化基因来自以前的非蛋白编码DNA序列,许多这类基因在雄性生殖结构中表达。在黑腹果蝇中,几个推定的新基因在精子发生过程中发挥了重要作用,但这些基因是否也能影响雄性以外的精子功能,目前还没有研究。我们发现了一个推定的新基因凯瑟琳-约翰逊(kj),它是雄性高水平生育能力所必需的。不表达 kj 的雄性能产生和转移正常储存在雌性体内的精子,但这些雄性的精子进入卵子的效率严重下降。利用标记转基因拯救构建体,我们观察到 KJ 蛋白在精子发生的不同阶段定位于核外围,但在成熟精子中检测不到。这些数据表明,KJ对精子的发育有影响,失去它就会导致受精能力下降。虽然之前的生物信息学分析表明 kj 基因仅限于黑腹果蝇,但我们在整个果蝇属中发现了具有保守同源关系、雄性偏向表达和预测蛋白特征的推定直向同源物,并在一些品系中发现了基因缺失的情况。因此,kj 有可能产生于果蝇的共同祖先,随后在黑腹果蝇中演化出重要作用。我们的研究结果展示了新基因进化所形成的雄性生殖的一个新方面,并为进一步研究果蝇精子进入卵子的机制提供了分子基础。
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引用次数: 0
Epigenetic heterogeneity shapes the transcriptional landscape of regional microglia 表观遗传异质性塑造了区域小胶质细胞的转录景观
Pub Date : 2024-08-09 DOI: 10.1101/2024.08.08.607229
Alexander Ve Margetts, Samara Jo Vilca, Florence Bourgain-Guglielmetti, Luis Miguel Tuesta
Microglia, the innate immune cells in the central nervous system, exhibit distinct transcriptional profiles across brain regions that are important for facilitating their specialized function. There has been recent interest in identifying the epigenetic modifications associated with these distinct transcriptional profiles, as these may improve our understanding of the underlying mechanisms governing the functional specialization of microglia. One obstacle to achieving this goal is the large number of microglia required to obtain a genome-wide profile for a single histone modification. Given the cellular and regional heterogeneity of the brain, this would require pooling many samples which would impede biological applications that are limited by numbers of available animals. To overcome this obstacle, we have adapted a method of chromatin profiling known as Cleavage Under Targets and Tagmentation (CUT&Tag-Direct) to profile histone modifications associated with regional differences in gene expression throughout the brain reward system. Consistent with previous studies, we find that transcriptional profiles of microglia vary by brain region. However, here we report that these regional differences also exhibit transcriptional network signatures specific to each region. Additionally, we find that these region-dependent network signatures are associated with differential deposition of H3K27ac and H3K7me3, and while the H3K27me3 landscape is remarkably stable across brain regions, the H3K27ac landscape is most consistent with the anatomical location of microglia which explain their distinct transcriptional profiles. Altogether, these findings underscore the established role of H3K27me3 in cell fate determination and support the active role of H3K27ac in the dynamic regulation of microglial gene expression. In this study, we report a molecular and computational framework that can be applied to improve our understanding of the role of epigenetic regulation in microglia in both health and disease, using as few as 2,500 cells per histone mark.
小胶质细胞是中枢神经系统中的先天性免疫细胞,在不同脑区表现出不同的转录特征,这对促进其专门化功能非常重要。最近,人们对鉴定与这些不同转录特征相关的表观遗传修饰产生了兴趣,因为这些修饰可能会增进我们对支配小胶质细胞功能特化的潜在机制的了解。实现这一目标的一个障碍是,获得单个组蛋白修饰的全基因组图谱需要大量小胶质细胞。鉴于大脑的细胞和区域异质性,这将需要汇集许多样本,这将阻碍受可用动物数量限制的生物学应用。为了克服这一障碍,我们采用了一种名为 "目标下裂解和标记(CUT&Tag-Direct)"的染色质剖析方法,来剖析与整个大脑奖赏系统中基因表达的区域差异相关的组蛋白修饰。与之前的研究一致,我们发现小胶质细胞的转录特征因大脑区域而异。然而,在这里我们报告说,这些区域差异也表现出了每个区域特有的转录网络特征。此外,我们还发现这些依赖于区域的网络特征与 H3K27ac 和 H3K7me3 的不同沉积有关,虽然 H3K27me3 的分布在不同脑区非常稳定,但 H3K27ac 的分布与小胶质细胞的解剖位置最为一致,这就解释了它们不同的转录特征。总之,这些发现强调了 H3K27me3 在细胞命运决定中的既定作用,并支持 H3K27ac 在小胶质细胞基因表达的动态调控中的积极作用。在这项研究中,我们报告了一个分子和计算框架,该框架可用于提高我们对小胶质细胞在健康和疾病中的表观遗传调控作用的理解,每个组蛋白标记只需使用 2500 个细胞。
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引用次数: 0
Genetic estimates and genome-wide association studies of antibody response in Tanzanian dairy cattle 坦桑尼亚奶牛抗体反应的遗传估计和全基因组关联研究
Pub Date : 2024-08-08 DOI: 10.1101/2024.08.05.606566
Luis E Hernandez-Castro, Elizabeth Anne Jessie Cook, Oswald Matika, Isaac Joseph Mengele, Shabani Kiyabo Motto, Shedrack Festo Bwatota, Bibiana Zirra-Shallangwa, Ricardo Pong-Wong, James Prendergast, Raphael Mrode, Philip G. Toye, Daniel Mushumbusi Komwihangilo, Eliamoni Lyatuu, Benedict E. Karani, Getrude Nangekhe, Okeyo Ally Mwai, Gabriel Mkilema Shirima, Barend Mark de Clare Bronsvoort
Identifying the genetic determinants of host defence against infectious pathogens is central to enhancing disease resilience and therapeutic efficacy in livestock. Here we have taken a genome-wide association approach to identify genetic variants associated with the presence of serological markers for important infectious diseases affecting dairy cattle in smallholder farms. Assessing 668,911 single-nucleotide polymorphisms in 1977 crossbreed cattle sampled from six regions of Tanzania, we identified high levels of interregional admixture and European introgression which may increase infectious disease susceptibility relative to indigenous breeds. Heritability estimates ranged from 0.03 (SE ± 0.06) to 0.44 (SE ± 0.07) depending on the pathogen assayed. Preliminary genome scans revealed several loci associated with seropositivity to the viral diseases Rift Valley fever and bovine viral diarrhoea, the protozoan parasites Neospora caninum and Toxoplasma gondii, and the bacterial pathogens Brucella sp, Leptospira hardjo and Coxiella burnetti. The associated loci mapped to genes involved in immune defence, tumour suppression, neurological processes, and cell exocytosis. We discuss future work to clarify the cellular pathways contributing to general and taxon-specific infection responses and to advance selective breeding and therapeutic target designs.
确定宿主防御传染性病原体的遗传决定因素对于提高牲畜的抗病能力和治疗效果至关重要。在此,我们采用了全基因组关联方法,以确定与影响小农农场奶牛的重要传染病血清学标记物存在相关的遗传变异。通过对来自坦桑尼亚六个地区的 1977 头杂交牛的 668,911 个单核苷酸多态性进行评估,我们发现了高水平的区域间混杂和欧洲引种,这可能会增加相对于本地品种的传染病易感性。遗传率估计值从 0.03(SE ± 0.06)到 0.44(SE ± 0.07)不等,取决于所测定的病原体。初步的基因组扫描发现了几个与病毒性疾病裂谷热和牛病毒性腹泻、原生动物寄生虫犬新孢子虫和弓形虫以及细菌性病原体布鲁氏菌、钩端螺旋体和烧伤克氏菌的血清阳性相关的基因位点。相关基因座映射到涉及免疫防御、肿瘤抑制、神经过程和细胞外泌的基因。我们讨论了今后的工作,以澄清导致一般和类群特异性感染反应的细胞途径,并推进选择性育种和治疗目标的设计。
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引用次数: 0
Using phylogenetic summary statistics for epidemiological inference 利用系统发生学汇总统计进行流行病学推断
Pub Date : 2024-08-07 DOI: 10.1101/2024.08.07.607080
Rafael C. Núñez, Gregory R. Hart, Michael Famulare, Christopher Lorton, Joshua T. Herbeck
Since the coining of the term phylodynamics, the use of phylogenies to understand infectious disease dynamics has steadily increased. As methods for phylodynamics and genomic epidemiology have proliferated and grown more computationally expensive, the epidemiological information they extract has also evolved to better complement what can be learned through traditional epidemiological data. However, for genomic epidemiology to continue to grow, and for the accumulating number of pathogen genetic sequences to fulfill their potential widespread utility, the extraction of epidemiological information from phylogenies needs to be simpler and more efficient. Summary statistics provide a straightforward way of extracting information from a phylogenetic tree, but the relationship between these statistics and epidemiological quantities needs to be better understood. In this work we address this need via simulation. Using two different benchmark scenarios, we evaluate 74 tree summary statistics and their relationship to epidemiological quantities. In addition to evaluating the epidemiological information that can be inferred from each summary statistic, we also assess the computational cost of each statistic. This helps us optimize the selection of summary statistics for specific applications. Our study offers guidelines on essential considerations for designing or choosing summary statistics. The evaluated set of summary statistics, along with additional helpful functions for phylogenetic analysis, is accessible through an open-source Python library. Our research not only illuminates the main characteristics of many tree summary statistics but also provides valuable computational tools for real-world epidemiological analyses. These contributions aim to enhance our understanding of disease spread dynamics and advance the broader utilization of genomic epidemiology in public health efforts.
自从提出系统动力学这一术语以来,利用系统发生学来了解传染病动态的情况稳步增加。随着系统动力学和基因组流行病学方法的增多和计算成本的增加,它们所提取的流行病学信息也在不断发展,以更好地补充通过传统流行病学数据所能了解到的信息。然而,要使基因组流行病学继续发展,并使不断积累的病原体基因序列发挥其潜在的广泛作用,从系统发生学中提取流行病学信息的工作就必须更加简单、高效。摘要统计提供了一种从系统发生树中提取信息的直接方法,但这些统计与流行病学数量之间的关系需要更好地理解。在这项工作中,我们通过模拟来满足这一需求。利用两种不同的基准方案,我们评估了 74 个系统树汇总统计量及其与流行病学数量之间的关系。除了评估可从每个汇总统计量推断出的流行病学信息外,我们还评估了每个统计量的计算成本。这有助于我们优化特定应用中汇总统计量的选择。我们的研究为设计或选择汇总统计的基本考虑因素提供了指导。通过开源 Python 库可以访问经过评估的汇总统计集以及其他对系统发育分析有帮助的函数。我们的研究不仅阐明了许多树状汇总统计的主要特点,还为现实世界的流行病学分析提供了宝贵的计算工具。这些贡献旨在加强我们对疾病传播动态的了解,并推动在公共卫生工作中更广泛地利用基因组流行病学。
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引用次数: 0
Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae 用于在酿酒酵母中进行 retron 介导的 CRISPR-Cas9 基因组编辑的改良载体
Pub Date : 2024-08-07 DOI: 10.1101/2024.08.06.606807
Tara N. Stuecker, Stephanie E. Hood, Julio Molina Pineda, Sonali Lenaduwe, Joshua Winter, Meru J. Sadhu, Jeffrey A. Lewis
In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas ‘cutting’ combined with homologous recombination of a ‘repair’ template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.
体内定点突变是一种强大的遗传工具,可用于测试特定等位基因在其正常基因组环境中的影响。虽然芽殖酵母拥有经典的定点诱变工具,但最近基于 CRISPR 的方法使用 Cas "切割 "结合 "修复 "模板的同源重组来引入所需的编辑,效率更高。然而,目前的方法仅限于完全原养的酵母菌株,而且依赖于效率相对较低的短 gRNA 克隆。因此,我们希望将 gRNA 及其同源修复模板顺式结合到单个寡核苷酸上,从而简化这一过程。此外,我们还希望利用一种新方法,即使用大肠杆菌重构子(EcRT)在体内扩增修复模板为多拷贝单链(ms)DNA,这是一种更有效的同源重组模板。为此,我们创建了一套表达 Cas9-EcRT 的质粒,只需一个步骤就能与 gRNA 修复模板质粒共同转化。我们的这套质粒包含不同的抗生素(Nat、Hyg、Kan)或辅助营养(HIS3、URA3)可选择性标记,可以编辑完全原营养的野生酵母菌株。除了传统的半乳糖诱导外,我们还生成了每种质粒的β-雌二醇诱导版本,以便于编辑在半乳糖上生长不良的酵母菌株。这种基于质粒的系统能以最少的步骤和时间实现 95% 的点突变编辑效率和 50% 的无标记缺失编辑效率。我们提供了如何使用该系统的详细分步指南。
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bioRxiv - Genetics
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