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.     

Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.     

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.     

用于分析微生物种类组成的微生物生态学研究方法

对环境中微生物群落进行分析对于理解该环境中各类微生物的功能具有重要意义,因此日益受到重视。用于环境中微生物群落分析的方法很多,将简要介绍基于PCR的研究方法,原位杂交技术,基因芯片技术,宏蛋白质组学技术等研究方法的原理、应用及其优缺点。     

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.     

微生物分子生态技术:16S rRNA/DNA方法

综述了以 1 6SrRNA/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 1 6SrRNA/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、 1 6SrDNA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。     

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。     

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.     

DegePrime,a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies

The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571–1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.     

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.     

A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations (>2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.