Multivariate Analysis of Complex DNA Sequence Electropherograms for High-Throughput Quantitative Analysis of Mixed Microbial Populations

High-throughput quantification of genetically coherent units (GCUs) is essential for deciphering population dynamics and species interactions within a community of microbes. Current techniques for microbial community analyses are, however, not suitable for this kind of high-throughput application. Here, we demonstrate the use of multivariate statistical analysis of complex DNA sequence electropherograms for the effective and accurate estimation of relative genotype abundance in cell samples from mixed microbial populations. The procedure is no more labor-intensive than standard automated DNA sequencing and provides a very effective means of quantitative data acquisition from experimental microbial communities. We present results with the Campylobacter jejuni strain-specific marker gene gltA, as well as the 16S rRNA gene, which is a universal marker across bacterial assemblages. The statistical models computed for these genes are applied to genetic data from two different experimental settings, namely, a chicken infection model and a multispecies anaerobic fermentation model, demonstrating collection of time series data from model bacterial communities. The method presented here is, however, applicable to any experimental scenario where the interest is quantification of GCUs in genetically heterogeneous DNA samples.     

A Rank-Based Sequence Aligner with Applications in Phylogenetic Analysis

Recent tools for aligning short DNA reads have been designed to optimize the trade-off between correctness and speed. This paper introduces a method for assigning a set of short DNA reads to a reference genome, under Local Rank Distance (LRD). The rank-based aligner proposed in this work aims to improve correctness over speed. However, some indexing strategies to speed up the aligner are also investigated. The LRD aligner is improved in terms of speed by storing -mer positions in a hash table for each read. Another improvement, that produces an approximate LRD aligner, is to consider only the positions in the reference that are likely to represent a good positional match of the read. The proposed aligner is evaluated and compared to other state of the art alignment tools in several experiments. A set of experiments are conducted to determine the precision and the recall of the proposed aligner, in the presence of contaminated reads. In another set of experiments, the proposed aligner is used to find the order, the family, or the species of a new (or unknown) organism, given only a set of short Next-Generation Sequencing DNA reads. The empirical results show that the aligner proposed in this work is highly accurate from a biological point of view. Compared to the other evaluated tools, the LRD aligner has the important advantage of being very accurate even for a very low base coverage. Thus, the LRD aligner can be considered as a good alternative to standard alignment tools, especially when the accuracy of the aligner is of high importance. Source code and UNIX binaries of the aligner are freely available for future development and use at http://lrd.herokuapp.com/aligners. The software is implemented in C++ and Java, being supported on UNIX and MS Windows.     

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.     

微生物分子生态技术：16SrRNA／DNA方法

综述了以16SrRNA／DNA为基础的分子生物学技术在环境微生物种群分析中的应用,目的是使相关的科研人员能够对16SrRNA／DNA技术有一个比较完整的了解,并可以开展初步的实验工作。主要内容包括：DNA指纹技术、16rDNA文库的建立、DNA测序及微生物分类鉴定(即系统发育树分析)、基因探针设计和测试、荧光原位杂交和核酸印迹杂交等。     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Cross-Kingdom Amplification Using Bacteria-Specific Primers: Complications for Studies of Coral Microbial Ecology

PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem.     

An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH₃, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).     

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.     

DNA序列信息的一种新的测度

根据信息理论给出了测度ＤＮＡ序列信息的一种新的方法,获得ＤＮＡ序列４个层次的信息量测度：Ｉｂ,Ｉｆ（１）,Ｉｆ（２）ａｎｄＩｆ（３）,这４种信息测度可分别用来测度ＤＮＡ的碱基序列、密码子序列、编码蛋白质序列和功能蛋白质序列的信息量。从Ｍ．ｅｄｕｌｉｓ的线粒体基因组中两个较短的编码蛋白质的ＤＮＡ序列和使用具有不同倍性的间并密码子组组成的模拟ＤＮＡ序列中所获得计算结果表明,这些信息测度确实能用来揭示所     

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

综合应用ITS及16SrDNA进行环境微生物生态研究

提出并介绍一种研究微生物生态的新方法.该方法将克隆测序和RISA图谱技术有机结合.其技术关键是PCR扩增的目标片段包含全长ITS和部分16S rDNA片段,既可利用16S rDNA进行系统发育分析,又可测定ITS片段大小并与RISA图谱进行比对定位.分析过程简单经济,易于操作,普通分子生物实验室即可实现.     

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S–23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants.     

DNA分子标记技术在生态学研究中的应用

分子生物学几种常见的分子标记技术如RFLP,RAPD,微卫星法,DNA序列分析使生态学研究从宏观步入了微观领域,极大的推动了生态学的发展,对这几种分子标记技术在生态学中的应用及其进展进行了综述,并比较了这些分子生物学技术各自的优缺点.     

A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.     

Microarray Applications in Microbial Ecology Research

Microarray technology has the unparalleled potential to simultaneously determine the dynamics and/or activities of most, if not all, of the microbial populations in complex environments such as soils and sediments. Researchers have developed several types of arrays that characterize the microbial populations in these samples based on their phylogenetic relatedness or functional genomic content. Several recent studies have used these microarrays to investigate ecological issues; however, most have only analyzed a limited number of samples with relatively few experiments utilizing the full high-throughput potential of microarray analysis. This is due in part to the unique analytical challenges that these samples present with regard to sensitivity, specificity, quantitation, and data analysis. This review discusses specific applications of microarrays to microbial ecology research along with some of the latest studies addressing the difficulties encountered during analysis of complex microbial communities within environmental samples. With continued development, microarray technology may ultimately achieve its potential for comprehensive, high-throughput characterization of microbial populations in near real time.

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