高通量测序诊断多重真菌与细菌感染所致重症肺炎1例

报道1例高通量测序诊断多重真菌与细菌感染性重症肺炎。患者男,67岁,咳嗽咳痰伴发热1周,胸闷2 d。体检:体温39℃,双肺呼吸音粗,可听及明显湿啰音及喘鸣音,未及胸膜摩擦音。实验室检验示白细胞、C-反应蛋白、降钙素原、血糖、血酮体及D-葡聚糖升高。肺部CT示双肺多发肺炎。痰液高通量测序联合病原学检查提示白念珠菌、曲霉菌、肺炎链球菌等多种真菌与细菌混合感染。诊断:重症肺炎。治疗:予利奈唑胺、替加环素联合伏立康唑抗感染,机械通气、补液、降糖、化痰等对症支持治疗。2周后体温恢复正常、病情好转,23 d后病愈出院。     

高通量测序技术在转座子研究中的应用

高通量测序技术极大地提高了测序效率,大幅度降低了测序成本,同时该技术具有特异性好、灵敏度高、精确性高等优势,目前已被广泛应用于遗传变异、转录组学和表观组学等研究。近年来,高通量测序技术也逐渐应用于转座子的研究,并取得了丰硕的成果。本文主要综述了高通量测序技术在转座子研究中的应用,包括转座子含量估算、靶点偏好性及分布、多态性及群体频率、稀有转座子的鉴定、转座子的水平转移以及转座子标签技术中的应用等,并简要介绍了目前研究中采用的主要测序策略和算法,及其存在的利弊和相应的解决方案。最后对高通量测序技术,尤其是第三代测序技术的发展趋势和它们在转座子未来的研究中的应用进行了展望,以期为相关的科研人员提供一个全面的了解和参考。     

Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project na?ve bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.     

棕榈科植物种子内生细菌群落多样性的高通量测序分析

【目的】探究棕榈科种子内生细菌群落组成及其随物种的差异。【方法】运用MiSeq高通量方法测定蒲葵、棕榈、加拿利海枣、山棕、软叶刺葵、短穗鱼尾葵及三药槟榔7种棕榈科种子内生细菌群落16S rRNA基因V3–V4区序列并进行生物信息学分析。【结果】7种测试种子中获得的V3–V4区有效序列数在2341–40671条之间,聚成97–590个操作分类单元(OTU),Shannon指数计算为1.35–2.94,以加拿利海枣种子最高,山棕种子最低;种群归类结果显示不同种子中测得的各级细菌分类阶层总数及组成不同,总丰度以厚壁菌门的肠球菌属最高,同为厚壁菌门的芽孢杆菌属次之;属级水平上,第一优势细菌属分别为:蒲葵种子(芽孢杆菌属,45.8%),棕榈种子(肠球菌属,51.2%),山棕种子(肠球菌属,12.1%),短穗鱼尾葵种子(肠球菌属,32.6%),三药槟榔种子(肠球菌属,42.9%),软叶刺葵种子(肠球菌属,28.3%),加拿利海枣种子(糖多孢菌属,31.2%)。各种子中次优势细菌属分别为:蒲葵种子(乳球菌属),棕榈、山棕及三药槟榔种子(类芽孢杆菌属),加拿利海枣种子(戈登氏菌属),软叶刺葵和短穗鱼尾葵种子(鞘脂单胞菌属);PICRUST基因预测显示各种子均包含多个与人体器官及人类疾病相关的KEGG功能模块。此外,各种子中均产生了丰度较高的外源物质降解、萜和聚酮合成、多糖合成等有益功能信息。【结论】棕榈科植物种子内生细菌群落多样性较为丰富,群落组成随物种不同而异。各种子内生细菌群体中普遍含多个与人和动物体相关的种群和功能信息,也定殖多种具有益功能性状的细菌类群,值得进一步研究。     

Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects.

Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.     

Bacterial genetics and molecular pathogenesis in the age of high throughput DNA sequencing

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高通量扩增子测序分析生防细菌对丹参根际和根表土壤真菌群落多样性的影响

利用高量测序方法探究生防细菌对丹参植株根际和根表土壤真菌群落多样性的影响。

向丹参植株根部施入生防细菌DS-R5,培养45 d后采集根际和根表土壤样品提取总DNA,扩增样品基因组DNA的V4―V5区后进行双端测序,利用生物信息学解析生防细菌对丹参植株根际和根表土壤真菌群落多样性的影响。

菌株DS-R5处理后增加了根际和根表土壤真菌群落的多样性和丰度;根际土壤共有物种种类大于根表土壤,说明菌株DS-R5处理后根际土壤处理与对照物种种类更接近,而对根表土壤中的微生物物种影响较大。真菌群落结构组成分析结果表明,不同土壤样品在门水平上共有优势真菌主要有子囊菌门、接合菌门、担子菌门和未分类;相比根表土壤对照样品,根表土壤处理样品中子囊菌门丰度下降了13.0%,接合菌门丰度升高了69.2%;根际土壤处理样品相比根际土壤对照样品,子囊菌门和接合菌门丰度分别升高了5.9%和8.9%,但二者差异无统计学意义。在属水平上,根表土壤样品经菌株DS-R5处理后提高了有益菌属的丰度,同时降低了有害菌属的丰度。

丹参植株施入生防细菌后,改变了根际土壤和根表土壤中微生物群落结构和多样性,本研究结果可以为利用生防细菌防控丹参根腐病提供理论参考。

     

Comparison of ITS1 and ITS2 rDNA in 454 sequencing of hyperdiverse fungal communities

Next-generation amplicon sequencing is a powerful tool in ecological studies of fungi. Technological development suggests that short fragment high-throughput techniques, e.g. Illumina, will gain importance in fungal community analyses. Thus there is a need for short (250 bp) and informative molecular identifiers. Here we compared ITS1 vs. ITS2 rDNA using empirical data from a study of hyperdiverse leaf-associated fungal communities. Our results suggest that ITS2 may be more variable and recovers more of the molecular diversity. We confirm an earlier in silico study showing that ITS1 and ITS2 yielded somewhat different taxonomic community compositions when blasted against public databases. However, we demonstrate that both ITS1 and ITS2 reveal similar patterns in community structure when analyzed in a community ecology context.     

High‐throughput environmental sequencing reveals high diversity of litter and moss associated protist communities along a gradient of drainage and tree productivity

Although previous studies, mostly based on microscopy analyses of a few groups of protists, have suggested that protists are abundant and diverse in litter and moss habitats, the overall diversity of moss and litter associated protists remains elusive. Here, high‐throughput environmental sequencing was used to characterize the diversity and community structure of litter‐ and moss‐associated protists along a gradient of soil drainage and forest primary productivity in a temperate rainforest in British Columbia. We identified 3262 distinct protist OTUs from 36 sites. Protists were strongly structured along the landscape gradient, with a significant increase in alpha diversity from the blanket bog ecosystem to the zonal forest ecosystem. Among all investigated environmental variables, calcium content was the most strongly associated with the community composition of protists, but substrate composition, plant cover and other edaphic factors were also significantly correlated with these communities. Furthermore, a detailed phylogenetic analysis of unicellular opisthokonts identified OTUs covering most lineages, including novel OTUs branching with Discicristoidea, the sister group of Fungi, and with Filasterea, one of the closest unicellular relatives to animals. Altogether, this study provides unprecedented insight into the community composition of moss‐ and litter‐associated protists.     

Ectomycorrhizal fungal communities of native and non-native Pinus and Quercus species in a common garden of 35-year-old trees

Non-native tree species have been widely planted or have become naturalized in most forested landscapes. It is not clear if native trees species collectively differ in ectomycorrhizal fungal (EMF) diversity and communities from that of non-native tree species. Alternatively, EMF species community similarity may be more determined by host plant phylogeny than by whether the plant is native or non-native. We examined these unknowns by comparing two genera, native and non-native Quercus robur and Quercus rubra and native and non-native Pinus sylvestris and Pinus nigra in a 35-year-old common garden in Poland. Using molecular and morphological approaches, we identified EMF species from ectomycorrhizal root tips and sporocarps collected in the monoculture tree plots. A total of 69 EMF species were found, with 38 species collected only as sporocarps, 18 only as ectomycorrhizas, and 13 both as ectomycorrhizas and sporocarps. The EMF species observed were all native and commonly associated with a Holarctic range in distribution. We found that native Q. robur had ca. 120% higher total EMF species richness than the non-native Q. rubra, while native P. sylvestris had ca. 25% lower total EMF species richness than non-native P. nigra. Thus, across genera, there was no evidence that native species have higher EMF species diversity than exotic species. In addition, we found a higher similarity in EMF communities between the two Pinus species than between the two Quercus species. These results support the naturalization of non-native trees by means of mutualistic associations with cosmopolitan and novel fungi.     

A 3-year study reveals that plant growth stage, season and field site affect soil fungal communities while cultivar and GM-trait have minor effects

In this three year field study the impact of different potato (Solanum tuberosum L.) cultivars including a genetically modified (GM) amylopectin-accumulating potato line on rhizosphere fungal communities are investigated using molecular microbiological methods. The effects of growth stage of a plant, soil type and year on the rhizosphere fungi were included in this study. To compare the effects, one GM cultivar, the parental isoline, and four non-related cultivars were planted in the fields and analysed using T-RFLP on the basis of fungal phylum specific primers combined with multivariate statistical methods. Additionally, fungal biomass and some extracellular fungal enzymes (laccases, Mn-peroxidases and cellulases) were quantified in order to gain insight into the function of the fungal communities. Plant growth stage and year (and agricultural management) had the strongest effect on both diversity and function of the fungal communities while the GM-trait studied was the least explanatory factor. The impact of cultivar and soil type was intermediate. Occasional differences between cultivars, the amylopectin-accumulating potato line, and its parental variety were detected, but these differences were mostly transient in nature and detected either only in one soil, one growth stage or one year.     

基于高通量测序分析盐角草根部内生细菌多样性及动态规律

【目的】探讨盐角草根部内生细菌群落多样性特征,揭示内生细菌群落结构在宿主关键发育期动态变化规律。【方法】通过罗氏454高通量测序获得内生细菌16S r RNA片段,然后进行生物信息分析。【结果】共获得20363条16S r RNA基因序列。各样品中可操作分类单元(operational taxonomic units,OTUs)在552–941之间。根部内生细菌群落主要包括4个门,其中Proteobacteri门占主导地位,其余依次是Firmicutes,Actinobacteria,Bacteroidetes。在Proteobacteria门中,Gammaproteobacteria是第一大纲,其后是Betaproteobacteria纲。宿主5个发育时期共同拥有7个细菌属,包括Azomonas,Serratia,Pantoea,Serpens,Pseudomonas,Halomonas,Kushneria。整体上看,Gammaproteobacteria纲在宿主5个发育时期呈现增长趋势。优势菌属在5个发育期存在差异,分别为Delftia,Kushneria,Serratia,Pantoea,Erwinia。所有文库总共含2108个特异OTUs,共同拥有5个相同OTUs。花期OTUs数量最多,结种期内生细菌多样性降低。在宿主的5个发育时期中,土壤p H、月均温和土壤盐含量这3个环境因子组成的集合对其内生细菌群落变化具有显著影响。【结论】盐角草内生细菌群落多样性丰富,宿主发育期决定了内生细菌群落结构。     

Climate and dispersal influence the structure of leaf fungal endophyte communities of Quercus gambelii in the eastern Great Basin,USA

Endophytic fungi form communities within living plant tissues. Their functions vary tremendously so the structures of their communities influence plant and ecosystem functions. We determined the separate effects of neutral and deterministic processes structuring communities of Quercus gambelii leaf endophytes by sampling north- and south-facing flanks of four canyons so local climate varied independently from spatial separation. We found that both deterministic (response to climate) and neutral (dispersal limitation) processes significantly influenced community structure. These findings, if applicable to other systems, may have consequences in both agricultural and natural ecosystems. Endophyte dispersal could limit agricultural production, ecosystem restoration or natural plant establishment. Thus deliberate inoculation may prove useful in some situations. Moreover, the influence of climate on fungal endophyte communities indicates that climate change may not only impact plant distributions directly, but may also do so indirectly via the effect of endophytes on plant tolerance to temperature and moisture extremes.     

Diversity and dynamics of microbial communities in brown planthopper at different developmental stages revealed by high‐throughput amplicon sequencing

The microbiome associated with brown planthopper (BPH) plays an important role in mediating host health and fitness. Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect. Here, we investigated the bacterial and fungal communities of BPH at different developmental stages using high‐throughput amplicon sequencing. Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development. The bacterial communities were generally richer than fungi in each developmental stage. At 97% similarly, 19 phyla and 278 genera of bacteria were annotated, while five fungal phyla comprising 80 genera were assigned. The highest species richness for the bacterial communities was detected in the nymphal stage. The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages. The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota, respectively. A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages, while the dominant fungal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage. Additionally, the microbial composition differed between male and female adults, suggesting that the microbial communities in BPH were gender‐dependent. Overall, our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.     

Ammonium inhibition through the decoupling of acidification process and methanogenesis in anaerobic digester revealed by high throughput sequencing

Objective

To reveal the shifts of microbial communities along ammonium gradients, and the relationship between microbial community composition and the anaerobic digestion performance using a high throughput sequencing technique.

Results

Methane production declined with increasing ammonium concentration, and was inhibited above 4 g l^?1. The volatile fatty acids, especially acetate, accumulated with elevated ammonium. Prokaryotic populations showed different responses to the ammonium concentration: Clostridium, Tepidimicrobium, Sporanaerobacter, Peptostreptococcus, Sarcina and Peptoniphilus showed good tolerance to ammonium ions. However, Syntrophomonas with poor tolerance to ammonium may be inhibited during anaerobic digestion. During methanogenesis, Methanosarcina was the dominant methanogen.

Conclusion

Excessive ammonium inhibited methane production probably by decoupling the linkage between acidification process and methanogenesis, and finally resulted in different performance in anaerobic digestion.