Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

采用Amplicon文库构建法对地中海贫血的基因检测

目的进一步完善现有地中海贫血的检测方法并且建立一种新的基于高通量测序的基因诊断方法,研究α/β地中海贫血基因的突变类型及分布情况。方法采用Amplicon文库构建法,基于高通量测序平台,对219例地中海贫血患者全血样本进行上机测序与数据分析。结果在219例地中海贫血患者中,共计检测出14种突变类型,包括CD122、CD125、CD142 3种非缺失型α-地贫突变及IVS-Ⅱ-654、CD41-42、CD17等11种常见β-地贫突变型别。此外,通过生物信息学软件作图分析,筛查出--^(SEA)、-α^(3.7)、-α^(4.2)3种缺失型α-地贫。结论本研究进一步完善了基于高通量测序的基因诊断方法,建立了一套Amplicon文库构建法,能准确、有效地用于诊断α/β地中海贫血基因的缺失与突变类型,对人群筛查,优生优育的遗传咨询,防止该病重症患儿的出生等具有重要意义。

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.

A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-liter rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

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 an-notated,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,re-spectively.A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages,while the dominant fun-gal 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 communties In BPH were gender-dependent.Uverall,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.

Metataxonomics of Internal Transcribed Spacer amplicons in cerebrospinal fluid for diagnosing and genotyping of cryptococcal meningitis

Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer(ITS)amplicons.Methods:The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls.Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital,Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018.ITS1 ribosomal deoxyribonucleic acid(rDNA)genes of 15 whole samples were amplified by universal forward primer ITS1(CTTGGTCATTTAGAGGAAGTAA)and reverse primer ITS2(GCTGCGTTCTTCATCGATGC),sequenced by Illumina MiSeq Benchtop Sequencer.The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples.Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis(PERMANOVA)in R software.Results:The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control.The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance(from 95.90%to 99.97%),followed by many other fungal groups(each<1.41%).ITS genotype was defined in 11 CSF samples,corresponding to ITS type 1,and confirmed by Sanger sequencing.A statistically significant difference(r2=0.65869,P=0.0014)between infectious group and control group was observed.Conclusions:The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples,which may provide a better diagnostic approach of cryptococcal infection.

Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford Nanopore Sequencing

Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background.To address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford Nanopore Prometh ION platform.This workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing,making it as low as 0.4/10,000 bp.In summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.

An integrative protocol for one‑step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.

Spatiotemporal dynamics of the microbial diversity on salt-preserved goatskins assessed by culturing and 16S rRNA gene amplicon sequencing

Wet-salted skin,as a special artificial high-salt environment,is rich in protein,fat,collagen and other nutrient substrates,and is a rich resource of halotolerant and halophilic microorganisms.However,knowledge gaps regarding the microbial community structure and inter taxa associations of wet-salted skin are large.In this study,the spatiotemporal dynamics and community structure of microorganisms present on wet-salted goatskins were investigated using 16S rRNA gene amplicon sequencing and culturable technique.Alpha diversity analysis based on Sobs,Chao,Ace and Shannon indices revealed that microbial diversity on the wet-salted goatskins exhibited a trend of‘down→up→down→flat’with time.During preservation,genera belonging to the bacteria domain such as Aci-netobacter,Weissella and Streptococcus were slowly dying out,whereas those belonging to halophilic archaea such as Natrialba and Haloterrigena were gradually flourishing.Moreover,to resist high-salt stress,microorganisms on the wet-salted goatskin gradually migrated from the outside to the inside,eventually leading to the microbial diversity inside the skin being the same as or even higher than that on the skin surface.Venn diagram analysis revealed that the strains of some genera,including Psychrobacter,Salimicrobium,Salinicola,Ornithinibacillus,Halomonas,Bacillus and Chromohalobacter,were distributed throughout the interior and exterior of the wet-salted goatskin and existed during various periods.Accordingly,45 protease-producing halophilic or halotolerant microorganisms were isolated and screened from the wet-salted goatskin using the gradient dilution plate method.Importantly,16S rRNA genes of some bacteria exhibited less than 99.5%similarity to valid published species,indicating that they likely are novel spe-cies and have a good potential for application.

Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis （Hemiptera： Aleyrodidae） adults using 16S rDNA amplicon pyrosequencing and FISH

Dialeurolonga malleswaramensis Sundararaj （Hemiptera： Aleyrodidae） is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene （mtCOl） helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswararnensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization （FISH） in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.

Catalog of operational taxonomic units and unified amplicon sequencing data for the microbiomes of medicinal plant roots

China has a rich history of cultivating medicinal plants,whose root microbial communities closely interact with the medicinal plants,thereby influencing their growth,health,and medicinal properties.Currently,researchers widely use 16S rRNA gene amplicon sequencing to study these root microbial communities.However,publicly available sequence datasets often lack essential sample information or contain errors,impeding the reuse of the datasets in the future.In this study,we aimed to create a united,reliable,and readily usable source of 16S rRNA gene sequences for medicinal plant root microbiomes.We compiled a catalog of 1392 microbiome samples for 58 medicinal plants from 58 studies,and manually provided essential sample information based on the experimental setup described in the associated papers.We then processed the sequences using a custom pipeline,generating a united catalog of operational taxonomic units(OTUs)and conducting taxonomic classification.We also pre-dicted the ecological functions of the communities for each sample.Finally,we used this dataset,to compare the rhizosphere bacterial communities of Pseudostellaria heterophylla from Fujian and Guizhou Provinces,revealing significant differences in the community composition of the same plant from different geographic locations.By providing a comprehensive and united catalog of amplicon sequences and OTUs for medicinal plant root bacterial communities,this study offers an invaluable resource for future comparative studies and data mining.

牛诺如病毒感染相关腹泻犊牛粪便菌群的特征分析

[目的]比较哺乳期健康犊牛和感染牛诺如病毒(Bovine norovirus,BNoV)的腹泻犊牛粪便菌群多样性和物种组成,初步揭示BNoV感染的腹泻犊牛粪便菌群特征。[方法]首先应用PCR和RT-PCR法对健康和腹泻犊牛粪便中的腹泻相关病原进行检测,然后采用16S rRNA扩增子测序技术对病原检测阴性的健康犊牛粪便(H组)和仅BNoV检测阳性的腹泻犊牛粪便(BNoV组)的微生物多样性、菌群组成和功能进行比较分析。[结果]与H组相比,BNoV组粪便菌群操作分类单元(OTUs)数量和Chao1指数显著升高(P<0.05),Shannon和Simpson指数极显著降低(P<0.01)。与H组相比,BNoV组粪便变形菌门、埃希-志贺属、丁酸球菌属丰度极显著升高(P<0.01),放线菌门、柯林斯菌属、巨球型菌属、厄氏菌属、双歧杆菌属、粪杆菌属等丰度极显著或显著降低(P<0.01;P<0.05)。双歧杆菌属、柯林斯菌属、巨球型菌属、厄氏菌属、粪杆菌属等在H组显著富集,而丁酸球菌属、埃希-志贺属、脆弱拟杆菌、梭菌属等在BNoV组显著富集。与H组相比,BNoV组粪便菌群胞内过程和信号传导、脂质代谢、新陈代谢、异生物质生物降解与代谢、萜类和聚酮类化合物代谢等显著或极显著上调(P<0.05;P<0.01),而膜转运、复制与修复、翻译、核苷酸代谢、遗传信息处理等显著或极显著下调(P<0.05;P<0.01)。[结论]感染BNoV的腹泻犊牛粪便菌群丰富度、多样性、物种组成和功能与健康犊牛显著不同,提示BNoV感染犊牛处于肠道菌群紊乱状态。

扩增子测序技术在中药分子鉴定中的应用

中药鉴定是保证中药用药安全的关键环节。中药分子鉴定通过分析遗传物质的差异实现鉴定,弥补了传统鉴定方法主观性较强的不足,成为中药鉴定方法的有力补充。扩增子测序技术通过对特定片段进行扩增,结合不同测序技术实现物种鉴定。文章对扩增子测序技术在生物鉴定中的优势、扩增子测序技术在中成药及中药材中的应用,以及扩增子测序技术在中药鉴定中的关键技术步骤进行总结梳理,分析扩增子测序技术在中药分子鉴定中的应用现状及未来发展,为后续中药分子鉴定工作提供研究思路。

Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled “Treated,” 0.15 g of glucose was added, and nothing was added to the container labeled “Control.” After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the “Treated” container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

纳米孔测序技术在实蝇类害虫检疫鉴定中的应用

【目的】纳米孔测序技术是单分子实时测序技术的一种,正在被广泛应用于临床快速诊断及微生物检测等领域。本研究以实蝇这一类重要的检疫性有害生物为例,探究该技术在昆虫检疫鉴定中应用的可行性,为昆虫检疫鉴定提供新方法。【方法】分别采用一代测序技术和纳米孔测序技术,对14种经形态学鉴定的实蝇成虫进行DNA条形码测序,通过BOLD和NCBI数据库对测序结果进行比对分析,并比较2种测序技术所得序列结果准确性的差异。【结果】通过纳米孔测序,14个实蝇样品在44 min内获得181Mb bases,每个样品平均得到11280条reads,单个reads的准确度为92.10%~94.53%;经一致性序列校正,所有实蝇样品均可得到与一代测序结果完全一致的序列,序列分析结果与形态学鉴定结果完全一致。【结论】采用本研究的实验流程和数据分析方法,纳米孔测序技术可以应用于实蝇类害虫的检疫鉴定,测序结果准确、高效;本研究提供的实验方案同样适用于基于扩增子测序的物种鉴定,满足大规模样品的高通量精准鉴定需求。

扩增子与宏基因组策略在猴痘病毒全基因组测序考核中的应用比较

目的比较扩增子与宏基因组测序在猴痘病毒全基因组测序考核中的应用效果,为猴痘病毒测序溯源与疫情防控提供技术参考。方法扩增子测序:对猴痘DNA先进行全基因组靶向扩增,再对扩增产物进行二代测序;宏基因组测序:对猴痘DNA直接进行二代测序。获得序列后使用CLC、IGV等软件分析2种不同测序方法的有效数据百分比、测序深度及不同测序深度下的病毒全基因组测序覆盖度等质量参数,评估测序质量。使用Nextclade进行病毒分型、序列突变及缺失情况分析,比较2种测序方法所获得序列的一致性及完整性。结果使用猴痘参比毒株MPXV-M5312_HM12_Rivers全基因序列(GenBank登录号:NC_063383.1)进行拼接后,扩增子测序和宏基因测序所获得有效数据百分比分别为99.72%和7.54%,测序深度范围分别为0×~334839×和44×~1000×,测序深度10×以上的位点覆盖度分别为90.3%和100%。通过IGV查看全基因组在不同测序深度下的覆盖情况发现,宏基因测序的全基因组位点测序深度覆盖均匀,而扩增子测序的全基因组位点测序深度覆盖不均、差异明显。病毒分型及序列一致性分析显示,2种方法所获得序列均为猴痘病毒IIb B.1分支,与参比序列相比宏基因测序结果存在73个突变位点,扩增子测序存在68个突变位点,深入分析发现扩增子测序有7个IIb B.1分支的共有突变位点未测到,还存在2个假阳性私有突变位点。结论在猴痘病毒全基因测序中可灵活使用扩增子测序或宏基因组测序方法,其中扩增子测序可以获得更多的有效数据量,而宏基因组测序在序列覆盖的均一性和准确性方面较好,本研究旨在为提高猴痘病毒全基因组测序成功率提供一点启发与技术参考。

扩增子测序技术在牡蛎GⅡ型诺如病毒基因多样性研究中的评估

牡蛎中诺如病毒基因多样的研究对病毒的流行传播和疫情防控具有十分重要的意义,二代测序技术(next generation sequencing, NGS)的扩增子测序具有高通量、高准确性等优势,被广泛应用于基因组学、转录组学和表观基因组学等领域。为评估扩增子测序技术对牡蛎中诺如病毒多样性研究的可行性,采用经典的巢式反转录PCR(reverse transcription-PCR, RT-PCR)方法对人为污染诺如病毒的牡蛎样品进行检测并将产物进行扩增子测序,对病毒基因型覆盖度和灵敏度进行分析。结果显示,在覆盖度方面,扩增子测序技术能够检测出人为添加到牡蛎中的6种GⅡ型诺如病毒,覆盖率达到100%;在灵敏度方面,对人为污染有不同数量诺如病毒的牡蛎进行检测及测序分析,证实扩增子测序的灵敏性较高,低于50个病毒粒子也能检测出。综上,扩增子测序技术提高了牡蛎中诺如病毒检测的准确性以及基因型的丰富度。

水华对中华绒螯蟹(Eriocheir sinensis)池塘养殖系统细菌群落及潜在致病菌的影响

近年来,水华频发制约着中华绒螯蟹养殖业的发展,环境微生物群落响应水华发生与消退机制是预防与治理水华的重要组成部分。为了解析中华绒螯蟹养殖系统细菌群落对水华的响应特征,采用16S rRNA基因扩增测序技术对拟浮丝藻水华下中华绒螯蟹养殖池塘细菌群落的组成、结构和潜在病原菌的变化进行了研究。结果显示,水体中的优势细菌类群为伯克霍尔德菌科(Burkholderiaceae)和微杆菌科(Microbacteriaaceae),底泥中的优势类群为Steroidobacteraceae菌科和Burkholderiaceae等;科水平下的蓝细菌门类群和其他大部分细菌群落呈显著正相关。养殖水体中潜在病原菌的丰度与水华的生消呈现出相反的趋势,OTU365和OTU1614(均属于Candidatus Similichlamydia epinepheli)是其主要的潜在病原菌,而在底泥中潜在致病菌表现出富集的特征,以OTU1280(大肠杆菌,Escherichia coli)为主。CCA结果显示,蓝藻暴发过程中,Tem、NO3--N、NO2--N及NH4+-N驱使水体蓝藻门细菌类群的变异,NO2--N是驱动养殖水体潜在致病菌群落变异的主导因子。研究结果为防治中华绒螯蟹养殖水华发生提供了宝贵的数据支撑。

基于环境RNA研究西太平洋浅水海山区真核微生物多样性及其与细菌的关系

海山遍布大洋海底,由于其特殊海底地形和水文动力环境,孕育了丰富且特殊的生物群落。采用环境RNA技术分析西太平洋M4浅水海山区真核微生物的多样性和分布,并与环境DNA技术检获的结果进行比较,揭示二者的异同。研究发现,RNA和DNA技术检获的表层和DCM层群落结构相似,在200 m以深的水层中群落结构差异较大。共甲藻纲的相对丰度在DNA数据中占比高于RNA数据,因此可能被基于eDNA的技术高估;而甲藻纲和金藻纲则相反,存在被低估的可能性。通过真核微生物与细菌的共现网络,揭示甲藻虽然是海山区叶绿素最大层共现网络中的关键类群,但其与细菌的相互关系弱,且代谢活性较弱。金藻中行混合营养的类群以及Bicoecea类群,在深层海水中普遍存在,且具有较高的代谢活性。环境RNA技术可检获有生命活性的物种分布,为海山区真核微生物多样性及分布提供更完整的认知。

45%二甲戊灵微胶囊悬浮剂对燕麦田土壤微生物群落的影响

【目的】研究45%二甲戊灵微胶囊悬浮剂对燕麦(Avena sativa)田土壤微生物群落的影响,为45%二甲戊灵微胶囊悬浮剂应用于燕麦田提供依据。【方法】用扩增子测序评价45%二甲戊灵微胶囊悬浮剂对土壤微生物群落结构的影响。【结果】燕麦田表层土壤微生物多样性评价结果显示:施用二甲戊灵30 d后土壤微生物观察到的物种(Observed species)、Chao1、Shannon和Simpson多样性指数与对照之间均无显著性差异。用多重响应排列程序、相似性分析和置换多元方差分析3种方法对测序结果进行主成分分析,结果显示:二甲戊灵对土壤微生物多样性无显著影响。在内蒙古农业大学农场试验点,二甲戊灵施用后节杆菌属(Arthrobacter)内微生物种群丰度明显降低;在乌兰察布市农林科学研究所试验田,二甲戊灵施用后真菌出壳多孢菌属(Stagonosporopsis)内微生物种群丰度显著降低,镰刀菌属(Fusarium)和短梗蠕孢属(Trichocladium)内微生物种群丰度显著升高。【结论】节杆菌属(Arthrobacter)、出壳多孢菌(Stagonosporopsis)、镰刀菌属(Fusarium)和短梗蠕孢属(Trichocladium)内微生物对二甲戊灵相对敏感,但45%二甲戊灵微胶囊悬浮剂不影响土壤微生物多样性指数。