Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses. html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5' terminus of the user-specified primer to the 3' terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.     

Microbial DNA profiling by multiplex terminal restriction fragment length polymorphism for forensic comparison of soil and the influence of sample condition

Aims:  To evaluate: (i) the impact of air-drying on bacterial, archaeal and fungal soil DNA profiles and (ii) the potential use of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) as a tool for forensic comparison of soil.
Methods and Results:  An M-TRFLP approach was used to profile bacterial, archaeal and fungal DNA profiles from five different soil sites. Air-drying soil significantly reduced the quantity of DNA but the number of operational taxanomic units (OTU) was unaffected. The impact of air-drying on soil DNA profiles was dependent on soil site and microbial primers. Fungal profiles were altered the least by air-drying. For prokaryotic profiles, air-drying altered the relative similarity/dissimilarity between soil sites. The M-TRFLP approach was more discriminatory compared with soil colour and single-taxa profiling, but did not significantly improve resolution between two similar soils.
Conclusions:  Of those tested, soil fungi were potentially the more robust target for application to soil forensic studies as they were altered less by air-drying and provided clear discrimination of soils from different sites. The M-TRFLP method demonstrated potential to achieve greater resolution, discriminating the soil sites based on both bacterial and fungal components.
Significance and Impact of the Study:  Soil DNA profiling has potential as a forensic tool, but sample condition and the appropriate selection of microbial target taxa must be considered.     

Residual polymerase activity-induced bias in terminal restriction fragment length polymorphism analysis

Residual activity of polymerase chain reaction DNA polymerases in restriction analyses strongly affected genetic profiling based on terminal restriction fragment length polymorphisms. Artificial fragment sizes produced as a result of 5'-overhang restriction site fill-in and addition of a terminal A may bias genetic profiling and genotyping of microbial communities. Efficient removal of polymerases retained original fragment sizes and significantly reduced this profiling bias in soil bacterial communities.     

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.     

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Monitoring of phytoplankton community structure using terminal restriction fragment length polymorphism (T-RFLP)

To establish molecular monitoring for the phytoplankton community in aquatic ecosystems, we analysed the terminal restriction fragment length polymorphism (T-RFLP) of small subunit ribosomal RNA gene (18S rDNA) sequences of nuclear genomes from the algal strains of culture collections and environmental samples of two freshwater reservoirs (Sangcheon reservoir and Seoho reservoir, Korea). Terminal restriction fragment (T-RF) length database was also constructed from twelve strains of algal culture collections to annotate and identify the phytoplankton species from T-RFLP profiles. Algal species in reservoirs were identified and monitored through the colony sequencing and T-RF length patterns of 18S rRNA. In this study, 41 unique clones were identified from two reservoirs including Chlorophyta, Cryptophyta, and Alveolata. In the case of Cryptomonas sp., we found significant linear relationships between T-RF peak areas and biovolumes by cell counting. Our results suggest that T-RFLP analysis can be a fast and quantitative monitoring tool for species changes in phytoplankton communities.     

Consensus multiplex PCR–restriction fragment length polymorphism (RFLP) for rapid detection of plant mitochondrial DNA polymorphism

A fast, simple, and efficient approach, termed consensus multiplex PCR–RFLP, was developed and employed to detect mitochondrial (mt)DNA variation in three orchid species, Spiranthes hongkongensis, S. sinensis , and S. spiralis . Using multiplex PCR, three pairs of consensus mitchondrial primers were added simultaneously into each reaction tube to amplify three nonoverlapping introns located in the NADH dehydrogenase genes. Fragment length differences in the multiplex PCR amplicons were directly detectable between S. spiralis and the other two species. Further restriction analysis of the multiplex PCR amplicons revealed sufficient mtDNA polymorphism, suitable for phylogenetic studies at the interspecific level. This approach is well suited for large-scale population surveys of mitochondrial genome diversity in plants. Additionally, the maternal mode of inheritance of organelle genomes renders this approach valuable for rapid identification of the origin and specific parentage of hybrid or allopolyploid species.     

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity.     

Effect of hydrogen on soil bacterial community structure in two soils as determined by terminal restriction fragment length polymorphism

The metabolism of hydrogen evolved from HUP^? legume nodules can alter bacterial community structures in the rhizosphere. Our earlier experiments demonstrated increased hydrogen uptake and appearance of white spots within bacterial colonies in H₂-treated soil. We were also able to isolate hydrogen-oxidizing bacteria from soil samples exposed to hydrogen, but not from samples exposed to air. To further understand the effect of hydrogen metabolism on soil microbial communities, in this study 16S rRNA terminal restriction fragment (TRF) profiles of different soil samples exposed to hydrogen gas under laboratory, greenhouse, and field conditions were analyzed. Relationships between soil bacterial community structures from hydrogen-treated soil samples and controls, illustrated by UPGMA (unpaired group mathematical averages) dendrograms, indicated a significant contribution of hydrogen metabolism to the variation in bacterial community. The intensity variation of TRF peaks includes both hydrogen-utilizing bacteria, whose growth were stimulated by hydrogen exposure, and other bacterial species whose growth was inhibited. Comparison of TRF profiles between laboratory and greenhouse samples showed that T-RFLP is a useful technique in the detection of root-related effects on soil bacterial community structure.     

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.     

Rapid DNA purification for restriction fragment length polymorphism analysis

This paper describes a method for isolation of DNA from blood samples involving a rapid chemical disintegration of proteins with 8 M urea and with a minimum of exposure to phenol. The DNA is further desalted and purified on Sephadex G-25 prepacked disposable columns. DNA isolated in this way was pure enough to be immediately cleaved by restriction enzymes.     

New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.     

TRFMA: a web-based tool for terminal restriction fragment length polymorphism analysis based on molecular weight

TRFMA provides a Web environment for analyzing T-RFLP results based on molecular weights of the fragments, rather than the numbers of nucleotides, to increase accuracy. The 16S rRNA data are saved as an XML file containing around 650 sequences (light version) and a MySQL database containing around 50 000 sequences (full version), which are connected to Web server via PHP5 and manipulated on an Internet browser. AVAILABILITY: TRFMA is freely available at http://myamagu.dent.kyushu-u.ac.jp/bioinformatics/trfma/index.html and can be downloaded from the same site.     

Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments

The distribution of bacterial communities terminal restriction fragment length polymorphism (T-RFLP) fingerprint patterns was evaluated at three proximal hydrocarbon-contaminated sites located within the harbour of Messina. In order to analyse the short-term variability of the individual terminal restriction fragment (T-RF) patterns, water samples were collected at the three sites on three occasions within 3 months (T(0), T(90) and T(91)). Four sample sizes, from 50 to 1000 ml for each collected sample, were analysed separately (36 total analysed samples) to evaluate the relationship between the sample size and the bacterial diversity estimates. The dominant T-RF groups mostly belonged to signatures of putative hydrocarbon-degrading bacteria, as revealed by the virtual analysis of the obtained bands. In order to test whether significant differences were occurring between the analysed samples, the Kruskal-Wallis non-parametric test was applied to the T-RF data set. Neither significant influence of the sample size nor short spatial variability within the three sampled sites was detected for each sampling time. On the contrary, significant temporal changes in the diversity of the bacterial communities were observed. These results were confirmed by the non-metric multidimensional scales (nMDS) analysis of the whole set of samples, which indicated three main groups corresponding to the three different sampling times. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring polluted marine environments, typically characterized by low diversity and high relative abundances of a few dominant groups.     

Diversity analysis of bacterial community compositions in sediments of urban lakes by terminal restriction fragment length polymorphism (T-RFLP)

Bacteria are crucial components in lake sediments and play important role in various environmental processes. Urban lakes in the densely populated cities are often small, shallow, highly artificial and hypereutrophic compared to rural and natural lakes and have been overlooked for a long time. In the present study, bacterial community compositions in surface sediments of three urban lakes (Lake Mochou, Lake Qianhu and Lake Zixia) in Nanjing City, China, were investigated using the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene and clone libraries. Remarkable differences in the T-RFLP patterns were observed in different lakes or different sampling stations of the same lake. Canonical correspondence analysis indicated that total nitrogen (TN) had significant effects on bacterial community structure in the lake sediments. Chloroflexi were the most dominant bacterial group in the clone library from Lake Mochou (21.7?% of the total clones) which was partly associated with its higher TN and organic matters concentrations. However, Bacteroidetes appeared to be dominated colonizers in the sediments of Lake Zixia (20.4?% of the total clones). Our study gives a comprehensive insight into the structure of bacterial community of urban lake sediments, indicating that the environmental factors played a key role in influencing the bacterial community composition in the freshwater ecosystems.