high-throughput sequencing

高通量测序
  • 文章类型: Journal Article
    对国家农业和食品研究组织的174种葡萄遗传资源进行了病毒学分析,日本。通过将葡萄藤分成6-10个植物的批次来制备总共20个批量样品。使用高通量测序分析每个批量样品,检测到27种病毒和5种类病毒,包括日本首次报道的六种病毒和一种类病毒(葡萄藤病毒F,L,T,小道消息KizilSapak病毒,葡萄藤西拉病毒1号,葡萄藤卫星病毒,和葡萄藤黄色斑点病毒2)。此外,检测到一种新型病毒病毒,其最大核苷酸序列同一性与其最接近的亲属仅为58%,葡萄病毒A(GVA)。这种新型病毒的基因组长度为7,461个核苷酸,编码五个开放阅读框,显示了病毒病毒的典型基因组结构。病毒的系统发育树将其置于基因组和推导的复制相关蛋白(RAP)和外壳蛋白(CP)的氨基酸中最接近GVA或葡萄病毒F(GVF)的不同位置。RAP和CP与GVA的氨基酸序列同一性,GVF,和其他病毒的最高53%和73%,分别,明显低于该属80%的物种划分阈值。低同一性和系统发育分析表明发现了一种暂时命名为葡萄病毒的新型病毒物种。
    Virome analysis was performed on 174 grape genetic resources from the National Agriculture and Food Research Organization, Japan. A total of 20 bulk samples was prepared by grouping the vines into batches of 6-10 plants. Each of the bulk samples was analyzed using high-throughput sequencing, which detected 27 viruses and 5 viroids, including six viruses and one viroid reported in Japan for the first time (grapevine viruses F, L, and T, grapevine Kizil Sapak virus, grapevine Syrah virus 1, grapevine satellite virus, and grapevine yellow speckle viroid 2). In addition, a novel vitivirus was detected with a maximum nucleotide sequence identity of only 58% to its closest relative, grapevine virus A (GVA). The genome of this novel virus was 7,461 nucleotides in length and encoded five open reading frames showing the typical genomic structure of vitiviruses. Phylogenetic trees of vitiviruses placed it in a distinct position nearest to GVA or grapevine virus F (GVF) in genomes and amino acids of deduced replication-associated protein (RAP) and coat protein (CP). The amino acid sequence identities of RAP and CP with GVA, GVF, and other vitiviruses were a maximum of 53% and 73%, respectively, which were significantly below the species demarcation threshold of 80% in the genus. The low identity and phylogenetic analyses indicate the discovery of a novel vitivirus species provisionally named grapevine virus P.
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  • 文章类型: Journal Article
    在分子水平上了解有机功能对于科学家揭示疾病机制和开发诊断或治疗方法很重要。
    本研究试图找到在11个大鼠器官中选择性表达的基因,包括肾上腺,大脑,结肠,十二指肠,心,回肠,肾,肝脏,肺,脾,脾和胃。
    将三只正常雄性Sprague-Dawley(SD)大鼠麻醉,他们上面提到的器官被收获,并提取新鲜器官中的RNA。使用Solexa高通量测序技术对纯化的RNA进行反转录和测序。基因的丰度通过每百万个碱基对测序的转录物序列的每千碱基片段的期望值(FPKM)来测量。寻找器官中具有最高表达水平的基因,并将其与器官中的中值进行比较。如果最高表达器官中的基因与中等表达器官中的基因显着不同(p<0.05),伴随着q值<0.05,占总丰度的70%以上,该基因被认为是器官中的选择性基因。
    京都基因和基因组百科全书(KEGG),和基因本体论(GO)通路被最高表达的基因富集。根据标准,筛选出1406个选择性基因,其中1,283在基因库中描述,其中123正在等待描述。已知的理解部分证实了器官中的KEGG和GO途径,并且大部分途径需要进一步研究。
    新的选择性基因和有机功能通路有助于科学家在分子水平上揭示器官的机制,和选择性基因\'产物是器官的候选疾病标记。
    UNASSIGNED: Understanding organic functions at a molecular level is important for scientists to unveil the disease mechanism and to develop diagnostic or therapeutic methods.
    UNASSIGNED: The present study tried to find genes selectively expressed in 11 rat organs, including the adrenal gland, brain, colon, duodenum, heart, ileum, kidney, liver, lung, spleen, and stomach.
    UNASSIGNED: Three normal male Sprague-Dawley (SD) rats were anesthetized, their organs mentioned above were harvested, and RNA in the fresh organs was extracted. Purified RNA was reversely transcribed and sequenced using the Solexa high-throughput sequencing technique. The abundance of a gene was measured by the expected value of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM). Genes in organs with the highest expression level were sought out and compared with their median value in organs. If a gene in the highest expressed organ was significantly different (p < 0.05) from that in the medianly expressed organ, accompanied by q value < 0.05, and accounted for more than 70% of the total abundance, the gene was assumed as the selective gene in the organ.
    UNASSIGNED: The Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) pathways were enriched by the highest expressed genes. Based on the criterion, 1,406 selective genes were screened out, 1,283 of which were described in the gene bank and 123 of which were waiting to be described. KEGG and GO pathways in the organs were partly confirmed by the known understandings and a good portion of the pathways needed further investigation.
    UNASSIGNED: The novel selective genes and organic functional pathways are useful for scientists to unveil the mechanisms of the organs at the molecular level, and the selective genes\' products are candidate disease markers for organs.
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  • 文章类型: Journal Article
    背景:来自植物样品的转录组数据通常包括许多源自RNA病毒基因组的读段,这些读段在RNA制备过程中同时分离。可以组装来自病毒的这些高通量测序读段以形成植物RNA基因组的新序列。
    结果:这里,我们鉴定了推定的新型有丝分裂病毒,通过葡萄砧木的高通量测序(HTS)对葡萄有丝分裂病毒1(GMV1)(Vitisspp.),并在RT-PCR分析中使用病毒特异性引物确认鉴定的病毒。GMV1的基因组RNA编码长度为2,496个核苷酸(nts)的完整开放阅读框(ORF)。由病毒基因组编码的RNA依赖性RNA聚合酶(RdRp)含有一个RdRp保守结构域。GMV1基因组的BLASTx分析显示与现有的有丝分裂病毒序列有33.18-56.75%的序列同一性。基于基因组序列的系统发育分析表明,GMV1与其他有丝分裂病毒聚集在不同的进化枝中。
    结论:葡萄1型有丝分裂病毒科的Unuamitovirus属中新发现的物种,靶向真菌线粒体。虽然大多数公认的有丝分裂病毒通常缺乏根据植物线粒体遗传密码的功能性RdRp,GMV1根据真菌和植物线粒体遗传密码编码完整的RdRp。
    BACKGROUND: Transcriptome data from a plant sample frequently include numerous reads originating from RNA virus genomes that were concurrently isolated during RNA preparation. These high-throughput sequencing reads from the virus can be assembled to form a new sequence for the plant RNA genome.
    RESULTS: Here, we identify putative novel mitovirus, grapevine mitovirus 1 (GMV1) through high-throughput sequencing (HTS) of grapevine rootstocks (Vitis spp.), and the identified virus was confirmed using virus-specific primers in RT-PCR assay. The genomic RNA of GMV1 encodes complete open reading frame (ORF) of 2,496 nucleotides (nts) in length. RNA-dependent RNA polymerase (RdRp) encoded by the viral genome contained one RdRp conserved domain. BLASTx analysis of GMV1 genome showed sequence identity of 33.18-56.75% with the existing mitovirus sequences. Phylogenetic analysis based on genome sequences showed that GMV1 clustered in a distinct clade to other mitoviruses.
    CONCLUSIONS: Grapevine mitovirus 1 represents a newly discovered species within the Unuamitovirus genus of the Mitoviridae family, targeting fungal mitochondria. While the majority of recognized mitoviruses typically lack a functional RdRp as per the plant mitochondrial genetic code, GMV1 encodes a complete RdRp in accordance with both fungal and plant mitochondrial genetic codes.
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  • 文章类型: Journal Article
    藻类大量繁殖威胁着全球水生生境的水质和生态系统稳定性,然而,调节浮游植物群落组装的动力学,开花的基础,在小水体中表现不佳。这里,我们采用高通量测序技术分析了连续12个月跨10个小水体的营养梯度构建浮游植物的驱动因素.蓝细菌和绿藻被确定为潜在的种子库,可引发花朵。鉴于蓝细菌占主导地位,在营养有限的水域中,群落组成的时间变化被抑制。环境因素和种间关系共同控制着时间浮游植物的动态。浮游植物,表现出更高的灵敏度,对环境和生物波动的反应比浮游细菌更快。这项研究提供了一个强大的基准标记,可以表征营养状态不同的小水体中的浮游演替轨迹。结果加强了支撑生物控制策略的生态机制,以减轻藻类的增殖并为这些无处不在的水生生态系统的水质管理提供信息。
    Algal blooms threaten water quality and ecosystem stability in aquatic habitats globally, yet dynamics regulating phytoplankton community assembly, the basis of blooms, remain poorly characterized in small water bodies. Here, we employed high-throughput sequencing to analyze drivers structuring phytoplankton across a trophic gradient of 10 small water bodies over 12 consecutive months. Cyanobacteria and Chlorophyta were identified as potential seed banks priming blooms. Temporal variation in community composition was muted in nutrient-limited waters given Cyanobacteria dominance. Environmental factors and interspecific relationships jointly governed temporal phytoplankton dynamics. Phytoplankton, exhibiting greater sensitivity, responded more rapidly than bacterioplankton to environmental and biological fluctuations. This research provides a robust bench mark characterizing planktonic successional trajectories across small water bodies varying in trophic status. Results reinforce ecological mechanisms underpinning biological control strategies to mitigate algal proliferation and inform water quality management of these ubiquitous aquatic ecosystems.
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  • 文章类型: Journal Article
    本研究旨在揭示细菌动力学对烤肉(Larou)品质和生物胺(BA)积累的影响。物理化学参数,游离氨基酸,BAs,氨基酸脱羧酶,并确定了微生物概况,在Larou成熟和储存过程中探索了它们的关系。结果表明,在拉罗成熟期,水分和亚硝酸钠显著降低(p<0.05),而pH值,NaCl,TBARS,挥发性碱性氮总量显著增加(p<0.05)。BAs主要在Larou的干熟和储存阶段形成,可能存在酪胺和苯乙胺中毒的风险。厚壁菌和放线菌是主要的门,优势属是葡萄球菌属,棒状杆菌和乳球菌。相关性分析显示棒状杆菌,短杆菌,乳酸菌,四球菌和葡萄球菌属。在确定Larou的质量和安全性方面发挥了至关重要的作用。
    在线版本包含补充材料,可在10.1007/s10068-023-01472-1获得。
    This study aimed to reveal the impact of bacterial dynamics on the quality and biogenic amine (BA) accumulation of dry-cured Chinese bacon (Larou). Physicochemical parameters, free amino acids, BAs, amino acid decarboxylase, and microbial profiles were determined, and their relationships were explored during Larou ripening and storage. The results showed that moisture and sodium nitrite decreased significantly during the Larou ripening stage (p < 0.05), while pH, NaCl, TBARS, and total volatile basic nitrogen considerably increased (p < 0.05). BAs were mainly formed during the stages of dry-ripening and storage of Larou and may present a risk of tyramine and phenylethylamine poisoning. Firmicutes and Actinobacteriota were the predominant phyla, and the dominant genera were Staphylococcus, Corynebacterium and Lactococcus. Correlation analysis showed Corynebacterium, Brevibacterium, Lactobacillus, Tetragenococcus and Staphylococci spp. played a crucial role in determining the quality and safety of Larou.
    UNASSIGNED: The online version contains supplementary material available at 10.1007/s10068-023-01472-1.
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  • 文章类型: Journal Article
    我们已经开发了一种用于转录因子(TF)的基因组足迹的新方法,该方法可以从核苷酸水平的DNase-seq数据中检测潜在的基因调控关系。我们引入了一种称为交联(XL)-DNase-seq的测定法,旨在捕获动态TFs的染色质相互作用。XL-DNase-seq中的温和交联步骤改善了动态TF的基于DNase的足迹的检测。足迹强度和可检测性取决于最佳的交联程序。这种方法可能有助于提取涉及以前无法检测的TFs的新基因调节回路。XL-DNase-seq方法在这里说明了激活的小鼠巨噬细胞样细胞,与炎性巨噬细胞有几个共同的特征。
    We have developed a novel method for genomic footprinting of transcription factors (TFs) that detects potential gene regulatory relationships from DNase-seq data at the nucleotide level. We introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. A mild cross-linking step in XL-DNase-seq improves the detection of DNase-based footprints of dynamic TFs. The footprint strengths and detectability depend on an optimal cross-linking procedure. This method may help extract novel gene regulatory circuits involving previously undetectable TFs. The XL-DNase-seq method is illustrated here for activated mouse macrophage-like cells, which share several features with inflammatory macrophages.
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  • 文章类型: Journal Article
    靶标下的切割和标签(CUT&Tag)提供高分辨率测序文库,用于分析不同的染色质组分。该协议详细说明了从新鲜或冷冻组织生成CUT&标签库的步骤。这个CUT&标记工作流程有九个主要步骤:从组织中分离细胞核,细胞核与刀豆蛋白A包被的珠子结合,一级抗体的结合,第二抗体的结合,结合pA-Tn5衔接子复合物,标签化,DNA提取,PCR,以及PCR后的清理和大小选择。该协议使我们能够在广泛的新鲜和冷冻组织类型中生成和测序CUT&标签库。
    Cleavage Under Targets and Tagmentation (CUT&Tag) provides high-resolution sequencing libraries for profiling diverse chromatin components. This protocol details the steps to generate CUT&Tag libraries from fresh or frozen tissues. This CUT&Tag workflow has nine main steps: isolation of nuclei from tissues, binding of nuclei to Concanavalin A-coated beads, binding of the primary antibody, binding of the secondary antibody, binding pA-Tn5 adapter complex, tagmentation, DNA extraction, PCR, and post-PCR cleanup and size selection. This protocol enabled us to generate and sequence CUT&Tag libraries across a broad range of fresh and frozen tissue types.
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  • 文章类型: Journal Article
    人类肠道微生物群在肾结石形成中的关键作用仍然未知。由于以前的测序技术的分类分辨率较低。因此,本研究旨在利用高通量测序技术探索肠道微生物群,以提供有价值的见解,并确定与肾结石形成有关的潜在细菌种类和代谢产物的作用。使用针对全长16SrRNA基因的PacBio测序检查了健康参与者和患者的整体肠道细菌群落及其潜在功能,加上石头和统计分析。大多数肾结石包括草酸钙和磷酸钙(75%),纯草酸钙(20%),和磷酸钙和磷酸镁(5%),Ca含量较高(130,510.5±108,362.7ppm),其次是P(18,746.4±23,341.2ppm)。在患者肾结石样本中发现微生物群落结构较弱,其次是病人的粪便样本,比健康参与者的粪便样本还要多。肾结石样本中最丰富的细菌种类是未培养的摩根氏菌,而在患者和健康参与者的粪便样本中,是类杆菌。同样,在物种水平上,Akkermansiamuciniphila在患者粪便样本中显著富集,而肾结石样本中拟杆菌的含量明显高于健康参与者的粪便样本。三种微生物代谢途径,TCA循环,脂肪酸氧化,和尿素循环,与健康参与者相比,肾结石患者显著富集。从物种水平推断细菌揭示了肾结石形成的关键参与者,增强肠道菌群的临床相关性。
    The critical role of the human gut microbiota in kidney stone formation remains largely unknown, due to the low taxonomic resolution of previous sequencing technologies. Therefore, this study aimed to explore the gut microbiota using high-throughput sequencing to provide valuable insights and identify potential bacterial species and metabolite roles involved in kidney stone formation. The overall gut bacterial community and its potential functions in healthy participants and patients were examined using PacBio sequencing targeting the full-length 16S rRNA gene, coupled with stone and statistical analyses. Most kidney stones comprised calcium oxalate and calcium phosphate (75%), pure calcium oxalate (20%), and calcium phosphate and magnesium phosphate (5%), with higher content of Ca (130,510.5 ± 108,362.7 ppm) followed by P (18,746.4 ± 23,341.2 ppm). The microbial community structure was found to be weaker in patients\' kidney stone samples, followed by patients\' stool samples, than in healthy participants\' stool samples. The most abundant bacterial species in kidney stone samples was uncultured Morganella, whereas that in patient and healthy participant stool samples was Bacteroides vulgatus. Similarly, Akkermansia muciniphila was significantly enriched in patient stool samples at the species level, whereas Bacteroides plebeius was significantly enriched in kidney stone samples than that in healthy participant stool samples. Three microbial metabolic pathways, TCA cycle, fatty acid oxidation, and urea cycle, were significantly enriched in kidney stone patients compared to healthy participants. Inferring bacteria at the species level revealed key players in kidney stone formation, enhancing the clinical relevance of gut microbiota.
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  • 文章类型: Journal Article
    四川酱味香肠的独特味道来自复杂的微生物代谢。微生物组成与不同风味成分之间的相关性尚未研究。该研究使用顶空固相微萃取作用与气相色谱质谱联用来寻找风味成分,并对16SrRNA进行高通量测序,以观察微生物群落的多样性和演替。相关网络模型预测了必需菌与风味开发之间的联系。研究表明,四川酱香香肠中的主要风味成分是酒精,醛类,和酯类。密切相关的微生物是明串珠菌,假单胞菌,嗜冷杆菌,黄杆菌,和Algoriella.微生物有助于生产各种风味化合物,如1-octen-3-ol,苯乙醛,己醛,(R,R)-2,3-丁二醇,和辛酸乙酯。这项工作增强了我们对四川酱香香肠发酵过程中细菌在风味开发中的多种功能的理解。
    The distinctive taste of Sichuan sauce-flavored sausage comes from an intricate microbial metabolism. The correlation between microbial composition and distinct flavor components has not been researched. The study used headspace solid-phase microextraction action with gas chromatography mass spectrometry to find flavor components and high-throughput sequencing of 16S rRNA to look at the diversity and succession of microbial communities. The correlation network model forecasted the connection between essential bacteria and the development of flavors. The study revealed that the primary flavor compounds in Sichuan sauce-flavored sausages were alcohols, aldehydes, and esters. The closely related microbes were Leuconostoc, Pseudomonas, Psychrobacter, Flavobacterium, and Algoriella. The microbes aided in the production of various flavor compounds, such as 1-octen-3-ol, benzeneacetaldehyde, hexanal, (R,R)-2,3-butanediol, and ethyl caprylate. This work has enhanced our comprehension of the diverse functions that bacteria serve in flavor development during the fermentation of Sichuan sauce-flavored sausage.
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  • 文章类型: Journal Article
    由于多种因素的综合作用,全球各地区出现新病毒的可能性增加,包括气候变化和国际贸易的扩大。植物病毒通过各种传播途径传播,包括众所周知的途径,如花粉,种子,和昆虫。然而,对这些已知机制之外的潜在传播途径的研究仍然有限。为了解决这个差距,这项研究采用了元转录组学分析来确定进口冷冻水果中植物病毒的存在,特别是樱桃和蓝莓。该分析旨在确定植物病毒可能引入国家的途径。病毒学分析显示,冷冻樱桃和蓝莓中存在六种植物病毒:樱桃病毒A(CVA),李子坏死环斑病毒(PNRSV),李子矮人病毒(PDV),prunus病毒F(PrVF),蓝莓休克病毒(BlShV),和蓝莓潜伏病毒(BlLV)。确定这些潜在的传播途径对于有效管理和防止植物病毒的传播和作物保护至关重要。这项研究强调了对冷冻水果采取强有力的质量控制措施和监测系统的重要性。强调需要采取积极措施来减轻与植物病毒潜在传播相关的风险。
    The possibility of new viruses emerging in various regions worldwide has increased due to a combination of factors, including climate change and the expansion of international trading. Plant viruses spread through various transmission routes, encompassing well-known avenues such as pollen, seeds, and insects. However, research on potential transmission routes beyond these known mechanisms has remained limited. To address this gap, this study employed metatranscriptomic analysis to ascertain the presence of plant viruses in imported frozen fruits, specifically cherries and blueberries. This analysis aimed to identify pathways through which plant viruses may be introduced into countries. Virome analysis revealed the presence of six species of plant viruses in frozen cherries and blueberries: cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), prunus virus F (PrVF), blueberry shock virus (BlShV), and blueberry latent virus (BlLV). Identifying these potential transmission routes is crucial for effectively managing and preventing the spread of plant viruses and crop protection. This study highlights the importance of robust quality control measures and monitoring systems for frozen fruits, emphasizing the need for proactive measures to mitigate the risk associated with the potential spread of plant viruses.
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