A comparison of DNA- and RNA-based clone libraries from the same marine bacterioplankton community

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by alpha-Proteobacteria in terms of repetitive DNA clones (52%), but gamma-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized if only the DNA or RNA libraries would have been analyzed alone. Of the DNA clones, 25.5% were found only in this library and no close relatives were detected in the RNA library. For clones from the RNA library, 21.5% of RNA clones did not indicate close relatives in the DNA library. Based on the comparisons between DNA and RNA libraries, our data indicate that the characterization of the bacterial community based on RNA has the potential to characterize distinct phylotypes from the marine environment, which remain undetected on the DNA level.     

Genome resource for the Indonesian coelacanth, Latimeria menadoensis

We have generated a BAC library from the Indonesian coelacanth, Latimeria menadoensis. This library was generated using genomic DNA of nuclei isolated from heart tissue, and has an average insert size of 171 kb. There are a total of 288 384-well microtiter dishes in the library (110,592 clones) and its genomic representation is estimated to encompass > or = 7X coverage based on the amount of DNA presumably cloned in the library as well as via hybridization with probes to a small set of single copy genes. This genomic resource has been made available to the public and should prove useful to the scientific community for many applications, including comparative genomics, molecular evolution and conservation genetics.     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.     

水环境细菌16S rNDA限制性片段长度多型性及群落结构分析

用细菌16S核菌体RNA基因（rDNA）限制性片段长度多型性描述了水环境细菌群落结构。从环境水样中直接分离DNA,以细菌特异的引物扩增16S rDNA。构建质粒文库,随机分离重组质粒,用限制性内切酶消化获得16S rDNA基因型,用基因型的种类及频率描述特定水体生境的细菌群落结构,该方法在分析水体隐含遗传多样性、揭示污染的生物学效应和评价水环境质量等方面具有重要的应用价值。     

Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat,Rattus norvegicus

Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescencein situhybridization were found to be chimeric, indicating a proportion of about 20–30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.     

A method for comparing multiple bacterial community structures from 16S rDNA clone library sequences

Culture-independent approaches, based on 16S rDNA sequences, are extensively used in modern microbial ecology. Sequencing of the clone library generated from environmental DNA has advantages over fingerprint-based methods, such as denaturing gradient gel electrophoresis, as it provides precise identification and quantification of the phylotypes present in samples. However, to date, no method exists for comparing multiple bacterial community structures using clone library sequences. In this study, an automated method to achieve this has been developed, by applying pair wise alignment, hierarchical clustering and principle component analysis. The method has been demonstrated to be successful in comparing samples from various environments. The program, named CommCluster, was written in JAVA, and is now freely available, at http://chunlab.snu.ac.kr/commcluster/.     

Construction of a bovine yeast artificial chromosome (YAC) library

We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research. We used leukocytes of a Japanese black bull ( Bos taurus ) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector. The library consists of 24 576 clones arranged in 256 96-well microtiter plates. An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb. The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36·4%. To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3·2 clones on average. In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.     

生物多样性数字图书馆系统的架构和实现

生物多样性研究工作急切需要一个建立在多源数据基础上的数字图书馆。基于虚拟用户社区的生物多样性数字图书馆除了在数据类型、存储需求、共享方式等方面具有一般数字图书馆的特点之外, 在数据挖掘和应用方面也有自己的一些特点。本文在对国内外数字图书馆调研和与生物多样性遗产图书馆(Biodiversity Heritage Library)及互联网档案(Internet Archive)项目的合作的基础上, 总结了各类数字图书馆中的数据类型, 对构建生物多样性数字图书馆相关的数据标准——Dublin Core和TaxonX作了简单介绍。然后设计了具有数据汇总、数据整理、转换和翻译以及数据对外服务三个模块的系统框架,提出了生物多样性数字图书馆的系统架构和功能,展示了已经实现的部分系统运行效果, 最后对今后在版权、全文识别、海量和扩展等方面的问题进行了讨论。     

A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library

The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10 010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper.     

A road less traveled by: exploring a decade of Ellman chemistry

The Ellman group has been one of the most influential in the development and widespread adoption of combinatorial chemistry techniques for biomedical research. Their work has included substantial methodological development for library synthesis with a particular focus on new scaffolds rationally targeted to biomolecules of interest and biologically relevant natural products. Herein we analyze a representative set of libraries from this group with respect to their biological and biomedical relevance in comparison to existing drugs and probe compounds. This analysis reveals that the Ellman group has not only provided new methodologies to the community but also provided libraries with unique potential for further biological study.     

Heteroduplex resolution using T7 endonuclease I in microbial community analyses

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.     

深圳大鹏湾海域锥状斯氏藻赤潮期间细菌群落结构变化研究

目的:近年来,赤潮在我国的发生呈增加的趋势,并造成了极大的经济损失。过去研究赤潮发生的机理主要集中在理化因素的影响,而越来越多的证据表明仅凭借营养盐等环境因素并不能解释大部分赤潮现象,藻际微生物可能发挥着重要作用。本文跟踪观测了2010年7月深圳大鹏湾海域爆发的锥状斯氏藻赤潮生消过程中细菌群落丰度种类的变化,从微生物与赤潮藻相互作用的角度探讨了赤潮的生消过程,讨论了不同时期不同关键菌群的特殊作用,为解释赤潮爆发和消亡提供了新的视角,为赤潮的监控和防治新方法的建立奠定了理论基础。方法:本文按时间顺序共采集该赤潮9次样本,利用末端限制性酶切片段长度多态性分析(T-RFLP)等分子生物学方法,通过主成分分析、克隆文库的构建,研究了微生物群落的变化过程,并探讨了特定种属的微生物在赤潮发生、发展和消亡过程中的作用。结果:从浮游细菌丰度来看,随着锥状斯氏藻细胞数量的波动,浮游细菌总数也随之呈现相应的变化。从浮游细菌的种类来看,它们主要属于变形杆菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)。从浮游细菌的动态变化过程来看,赤潮生消过程中,浮游细菌群落呈现出一定的演替现象,特别是在赤潮后期,群落主成分变化尤为显著。从关键菌群的作用来看,属于γ变形杆菌门的Alteromonas sp.一直占有较高的丰度,赤潮中后期受到菌群相互作用导致比例下降,而赤潮后期其他关键菌群的丰度的增高可能是导致赤潮消亡的重要原因。结论:本文利用T-RFLP这一DNA指纹技术分析微生物群落结构和多样性特征,通过研究赤潮生消过程中藻际浮游细菌群落的动态变化,发现随着赤潮的发展,浮游细菌群落发生着相应的变化。结果说明赤潮藻体丰度数量的改变影响着浮游细菌群落的组成。相对地,细菌群落的适应调整迅速造成赤潮藻体局部生长环境的改变,从而影响赤潮的发展过程。     

A fungal mock community control for amplicon sequencing experiments

Microbial ecology has been profoundly advanced by the ability to profile complex microbial communities by sequencing of marker genes amplified from environmental samples. However, inclusion of appropriate controls is vital to revealing the limitations and biases of this technique. “Mock community” samples, in which the composition and relative abundances of community members are known, are particularly valuable for guiding library preparation and data processing decisions. I generated a set of three mock communities using 19 different fungal taxa and demonstrate their utility by contrasting amplicon sequencing data obtained for the same communities under modifications to PCR conditions during library preparation. Increasing the number of PCR cycles elevated rates of chimera formation, and of errors in the final data set. Extension time during PCR had little impact on chimera formation, error rate or observed community structure. Polymerase fidelity impacted error rates significantly. Despite a high error rate, a master mix optimized to minimize amplification bias yielded profiles that were most similar to the true community structure. Bias against particular taxa differed among ITS1 vs. ITS2 loci. Preclustering nearly identical reads substantially reduced error rates, but did not improve similarity to the expected community structure. Inaccuracies in amplicon sequence‐based estimates of fungal community structure were associated with amplification bias and size selection processes, as well as variable culling rates among reads from different taxa. In some cases, the numerically dominant taxon was completely absent from final data sets, highlighting the need for further methodological improvements to avoid biased observations of community profiles.     

Fly-TILL: Reverse genetics using a living point mutation resource

Mutagenesis with ethylmethanesulfonate (EMS) has been the standard for traditional genetic screens, and in recent years has been applied to reverse genetics. However, reverse-genetic strategies require maintaining a viable germline library so that mutations that are discovered can subsequently be recovered. In applying our TILLING (Targeting Induced Local Lesions IN Genomes) method to establish a Drosophila reverse-genetic service (Fly-TILL), we chose to screen the Zuker lines, a large collection of EMS-mutagenized second- and third-chromosome balanced lines that had been established for forward-genetic screening. For the past four years, our Fly-TILL service has screened this collection to provide ~150 allelic series of point mutations for the fly community. Our analysis of >2000 point mutations and indels has provided a glimpse into the population dynamics of this valuable genetic resource. We found evidence for selection and differential recovery of mutations, depending on distance from balancer breakpoints. Although this process led to variable mutational densities, we have nevertheless been able to deliver valuable mutations in genes selected by Fly-TILL users. We anticipate that our findings will help guide the future implementation of point-mutation resources for the Drosophila community.     

Comparing sediment bacterial communities in the macrophyte-dominated and algae-dominated areas of eutrophic Lake Taihu, China

Bacterial community structure and the effects of several environmental factors on bacterial community distribution were investigated in the sediment of the macrophyte-dominated and algae-dominated areas in a large, shallow, eutrophic freshwater lake (Lake Taihu, China). Surface sediment samples were collected at 6 sampling sites (3 sites from each of the 2 areas) on 15 February and 15 August 2009. Based on cluster analysis of the DGGE banding patterns, there were significant seasonal variations in the structure of the sediment bacterial community in the macrophyte- and algae-dominated areas, and site-specific variation within an area and between 2 areas. However, there were no significant between-area variations due to the large within-area variation. Analysis of DNA sequences showed that there were differences in the species composition of the sediment bacteria between the macrophyte- and algae-dominated area clone libraries. In the macrophyte-dominated area library, the bacterial community was dominated by Deltaproteobacteria, Verrucomicrobia, Acidobacteria, Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. OP10 was found in the library of this area but not in the algae-dominated area library. The algae-dominated area library was dominated by Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Acidobacteria. Cyanobacteria, Alphaproteobacteria, and Planctomycetes were found in this area library but not in the macrophyte-dominated area library. Canonical correspondence analysis demonstrated that total phosphorus and water temperature were the dominant environmental factors affecting bacterial community composition in the sediment.     

Bacterial diversity of metagenomic and PCR libraries from the Delaware River

To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.     

Construction of a BAC library and generation of BAC end sequence-tagged connectors for genome sequencing of the African malaria mosquito Anopheles gambiae

A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles gambiae, the major human malaria vector in sub-Saharan Africa, was constructed and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in eighty 384-well plates. The estimated average insert size of the library is 133 kb, with an overall genome coverage of approximately 14-fold. The ends of approximately two-thirds of the clones in the library were sequenced, yielding 32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR screening of the library indicated a random distribution of BACs in the genome, although one gap encompassing the white locus on the X-chromosome was identified. Furthermore, combined with another previously constructed BAC library (ND-1), ~2,000 BACs have been physically mapped by polytene chromosomal in situ hybridization. These BAC end pair mates and physically mapped BACs have been useful for both the assembly of a fully sequenced A. gambiae genome and for linking the assembled sequence to the three polytene chromosomes. This ND-TAM library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/ and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.     

Effects of field-grown genetically modified Zoysia grass on bacterial community structure

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P?0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.