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.     

Horizontal Heterogeneity of Denitrifying Bacterial Communities in Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis

Although it is widely believed that horizontal patchiness exists in microbial sediment communities, determining the extent of variability or the particular members of the bacterial community which account for the observed differences among sites at various scales has not been routinely demonstrated. In this study, horizontal heterogeneity was examined in time and space for denitrifying bacteria in continental shelf sediments off Tuckerton, N.J., at the Rutgers University Long-Term Ecosystem Observatory (LEO-15). Characterization of the denitrifying community was done using PCR amplification of the nitrous oxide reductase (nosZ) gene combined with terminal restriction fragment length polymorphism analysis. Spatial scales from centimeters to kilometers were examined, while temporal variation was assayed over the course of 1995 to 1996. Sorenson's indices (pairwise similarity values) were calculated to permit comparison between samples. The similarities of benthic denitrifiers ranged from 0.80 to 0.85 for centimeter scale comparisons, from 0.52 to 0.79 for meter level comparisons, and from 0.23 to 0.53 for kilometer scale comparisons. Sorenson's indices for temporal comparisons varied from 0.12 to 0.74. A cluster analysis of the similarity values indicated that the composition of the denitrifier assemblages varied most significantly at the kilometer scale and between seasons at individual stations. Specific nosZ genes were identified which varied at centimeter, meter, or kilometer scales and may be associated with variability in meio- or macrofaunal abundance (centimeter scale), bottom topography (meter scale), or sediment characteristics (kilometer scale).     

Statistical Assessment of Variability of Terminal Restriction Fragment Length Polymorphism Analysis Applied to Complex Microbial Communities

The variability of terminal restriction fragment polymorphism analysis applied to complex microbial communities was assessed statistically. Recent technological improvements were implemented in the successive steps of the procedure, resulting in a standardized procedure which provided a high level of reproducibility.Terminal restriction fragment length polymorphism (T-RFLP) analysis is a robust, high-resolution, high-throughput, rapid, and cost-effective method for studying the structures of microbial communities (3, 10). T-RFLP analysis is based on group-specific variations in the restriction patterns of molecular markers essential to all life forms (i.e., rRNA genes) or unique to a particular physiological group (e.g., ammonia-oxidizing and sulfate-reducing bacteria) which generate specific and characteristic terminal restriction fragment (T-RF) patterns from mixed fluorescently labeled amplicon pools of environmental nucleic acid extracts. This analysis has developed recently into one of the favorite techniques for the rapid assessment of the structures of bacterial communities. Refinements of the technique and data analysis have been introduced (5, 8, 11, 14, 20-22). Improvements have been made to the sampling procedure (16), to the DNA extraction and amplification steps (17, 19, 26), and to enzymatic restriction digestion (2, 6). Statistical analysis has also been improved in the treatment of the raw data and the selection of logical binning and clustering algorithms resulting, for instance, in the alignment of replicate profiles into a single consensus profile (1, 13). Finally, recent developments have been proposed for the statistical analysis of the profiles using multivariate techniques from numerical ecology (4, 7, 9, 23-25, 27).Both the resolution and reproducibility of T-RFLP analysis have already been assessed using artificially created bacterial communities (12) comprising up to 30 different clones or bacterial species. However, to the best knowledge of the authors, so far no study has been conducted to assess statistically the dissimilarities obtained in the electropherogram profiles when more complex bacterial communities from natural samples have been analyzed. The main purpose of this report is then to assess statistically the resolution and reproducibility of a standardized T-RFLP protocol, as applied to the analysis of 16S rRNA gene pools from complex communities. The statistical analysis was carried out at successive steps of the procedure, from the initial PCR amplification to the sizing of the obtained T-RFs.The samples used for this study were taken from a sequencing batch bubble column reactor inoculated with activated sludge from a municipal wastewater treatment plant and operated in such a way as to produce aerobic granular sludge able to remove carbon, nitrogen, and phosphate from an artificial wastewater sample containing acetate, ammonium, and phosphate. Samples were taken at different steps of operation of the reactor systems. The standardized protocol used in the present report is presented in detail in the supplemental material. Note that the methodology implied in the extraction of the total bacterial DNA is not discussed in the context of this work. The T-RFLP protocol was conceived on the basis of recent developments made in the protocol at various stages of the T-RFLP analysis and was implemented with optimized procedures allowing us to minimize potential biases and to ensure a high degree of reproducibility. Whenever possible, technological advances in instrumentation were included, as for instance with the application of optimized electrophoresis conditions and the use of more complex sizing standards and brighter fluorochromes. The use of relatively large and precise amounts of digested PCR fragments (200 ng per replica) also contributed to a drastic reduction of the background noise, which was usually observed to be equal to only about 10 relative fluorescence units (RFU).Numerical treatment and analysis of the data were carried out with R (R Development Core Team) and the Vegan library (18). We used asymmetric dissimilarity indices to compare T-RFLP profiles using the Jaccard formula, so that the double absence of a T-RF was not considered a resemblance between two profiles (15). The Jaccard dissimilarity was applied to binary data, i.e., the presence/absence of T-RFs. Moreover, to take into account the relative intensity of T-RF areas within each profile in the comparison, we used Ruzicka dissimilarity, which is the Jaccard index applied to quantitative data. Both dissimilarity measures range from 0 (identical profiles) to 1 (different profiles with no T-RF in common). Numerical treatment of the data was also carried out on the modified results, so as to reduce potential biases induced by the inconsistent presence of T-RFs showing very small amounts of fluorescence. T-RF signals just above the detection threshold (low signal-to-noise ratio) can be a cause of suboptimal fingerprinting reproducibility. For this reason, small-area T-RFs (<300 RFU) were suppressed when they were not present in all replicate profiles of a sample.     

Terminal Restriction Fragment Length Polymorphism Analysis of Soil Bacterial Communities under Different Vegetation Types in Subtropical Area

Soil microbes are active players in energy flow and material exchange of the forest ecosystems, but the research on the relationship between the microbial diversity and the vegetation types is less conducted, especially in the subtropical area of China. In this present study, the rhizosphere soils of evergreen broad-leaf forest (EBF), coniferous forest (CF), subalpine dwarf forest (SDF) and alpine meadow (AM) were chosen as test sites. Terminal-restriction fragment length polymorphisms (T-RFLP) analysis was used to detect the composition and diversity of soil bacterial communities under different vegetation types in the National Natural Reserve of Wuyi Mountains. Our results revealed distinct differences in soil microbial composition under different vegetation types. Total 73 microbes were identified in soil samples of the four vegetation types, and 56, 49, 46 and 36 clones were obtained from the soils of EBF, CF, SDF and AM, respectively, and subsequently sequenced. The Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant in all soil samples. The order of Shannon-Wiener index (H) of all soil samples was in the order of EBF>CF>SDF>AM, whereas bacterial species richness as estimated by four restriction enzymes indicated no significant difference. Principal component analysis (PCA) revealed that the soil bacterial communities’ structures of EBF, CF, SDF and AM were clearly separated along the first and second principal components, which explained 62.17% and 31.58% of the total variance, respectively. The soil physical-chemical properties such as total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) were positively correlated with the diversity of bacterial communities.     

Comparison of Bacterial Communities in New England Sphagnum Bogs Using Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Wetlands are major sources of carbon dioxide, methane, and other greenhouse gases released during microbial degradation. Despite the fact that decomposition is mainly driven by bacteria and fungi, little is known about the taxonomic diversity of bacterial communities in wetlands, particularly Sphagnum bogs. To explore bacterial community composition, 24 bogs in Vermont and Massachusetts were censused for bacterial diversity at the surface (oxic) and 1 m (anoxic) regions. Bacterial diversity was characterized by a terminal restriction fragment length (T-RFLP) fingerprinting technique and a cloning strategy that targeted the 16S rRNA gene. T-RFLP analysis revealed a high level of diversity, and a canonical correspondence analysis demonstrated marked similarity among bogs, but consistent differences between surface and subsurface assemblages. 16S rDNA sequences derived from one of the sites showed high numbers of clones belonging to the Deltaproteobacteria group. Several other phyla were represented, as well as two Candidate Division-level taxonomic groups. These data suggest that bog microbial communities are complex, possibly stratified, and similar among multiple sites.     

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.     

末端限制性片段长度多态性(T-RFLP)技术的系统误差分析

T-RFLP技术是一种新近发展起来的分析微生物多样性的分子生物学方法,与其它多样性分析技术相比,具有一些不可比拟的优势,但T-RFLP技术操作流程将对结果产生系统误差的程度鲜有报道。实验以紫茎泽兰入侵过程中4种土样中的nifH基因多样性分析为例,进行了只改变T-RFLP操作流程中一个步骤的3次重复分析,结果表明:限制性内切酶种类对T-RFLP分析结果的可重复性影响最大,PCR次之,而毛细管电泳对结果的可重复性几乎没有影响。     

Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

末端限制性片段长度多态性技术分析硝化细菌微生物多样性

利用T_RFLP(末端限制性片段长度多态性)技术,分析硝化细菌富集反应器中的微生物群落结构,并对硝化细菌的丰度进行半定量研究。结果表明,培养48h后,硝化细菌富集效果最佳,多样性指数与初始培养相比下降了62.80%,富集出的硝化细菌主要为亚硝酸盐氧化菌(Nitrobacter)。同时对投加该硝化细菌前后的对虾养殖水体进行微生物多样性的动态研究,并推测了虾塘水中可能稳定存在的几种主要细菌种类,其中投加富集硝化细菌前后均存在的细菌种类包括短芽孢杆菌Brevibacillus brevis、微杆菌Microbacterium lactium、固氮弧菌Azoarcus indigens或者霍氏鲍特菌Bordetella holmesii。     

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.     

Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.     

Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.Terminal restriction fragment length polymorphism (TRFLP) profiling was originally developed as a means of genotyping mixed DNA samples (30) and is currently being employed in fungal community ecology studies (3, 5, 6, 7, 10, 13, 19, 22, 26, 27, 29, 33, 38), despite a number of technical and conceptual difficulties (11). Briefly, TRFLP profiling involves amplifying the DNA in pools of mixed genetic material with fluorescently labeled primers, digesting the products with restriction endonucleases, and sizing the labeled terminal fragments in a sequencer. The difference in the positions at which the different restriction enzymes cleave DNA is thought to provide enough variability for such DNA mixtures to be characterized and the contributing organisms to be identified.However, the technique is not without its problems. DNA extraction and PCR amplification biases burden most modern molecular techniques, including TRFLPs (18, 25). Additionally, concerns exist regarding the ability of the differences between primer-enzyme pairs (PEPs) to generate sufficiently different fragment sizes (2), the success of enzymatic cleavage (2), the dependency on the detection threshold of the sequencer (4), and the accuracy of DNA sizing (1). The choice of the primer pairs and restriction enzymes to be used has also been a matter of concern since the appearance of TRFLP profiling. Liu et al. (30) performed virtual digestion of all the bacterial RNA sequences in the Ribosomal Database Project database (release V) with 10 different enzymes and four primer pairs. This pioneering work showed the importance of avoiding enzymes with highly conserved target motifs, something that later became recognized as a major source of TRFLP bias (2, 14, 16, 32). Similar studies have been performed by Osborn et al. (36), Dunbar et al. (12), Engebretson and Moyer (15), and Cardinale et al. (8).The first virtual TRFLP analysis involving a database of fungal DNA sequences was performed by Edwards and Turco (14). This consisted of virtual digestion, by use of six restriction endonucleases, of 316 internally transcribed spacer (ITS) sequences belonging to a number of ectomycorrhizal genera. Avis et al. (2) found only small differences in the diversity of the TRFLPs produced in silico by three PEPs when using their own fungal ITS database, although these differences increased with sample number in iterative analysis. Recent advances using automated resources, such as REPK software (9), have allowed optimal enzyme selection for TRFLP profiling of previously defined communities of organisms. This software selects up to four restriction endonucleases capable of discriminating a desired number of sequence groups. However, this system relies on a priori information, which in real biological communities may not available.The aim of the present work was to improve selection of restriction enzymes for use in the TRFLP profiling of the ITS sequences of unknown fungal communities.     

Interpreting Ecological Diversity Indices Applied to Terminal Restriction Fragment Length Polymorphism Data: Insights from Simulated Microbial Communities

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E_var) and true Shannon diversity (H′), with correlations between 0.71 and 0.81. TRF-H′ and true H′ were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H′ cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E_var). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.     

The Use of Terminal Restriction Fragment Length Polymorphism (T-RFLP) for the Characterisation of Microbial Communities in Marine Sediments

Terminal Restriction Fragment Length Polymorphism (T-RFLP) of PCR amplified 16S rRNA genes was used to investigate microbial communities in the sediments of Ria Formosa, Portugal. Five replicates of surface sand sediments were collected at an artificial inlet to the sea, between June 2001 and July 2002. Restriction enzymes Msp1 and Hha1 provided 57 different terminal fragments (T-RFs). The sediments were essentially dominated by the same ribotypes throughout the year, with seasonal shifts attributed to minor ribotypes. Principal component analysis of the T-RFs profile revealed no consistent pattern of temporal variation and no consistent grouping of replicate sediment samples. The results suggest that the small-scale spatial variability outweighs the seasonal variability. Phylogenetic affiliations suggested that the dominant bacteria were representatives of the α-Proteobacteria group.     

TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets

We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.     

Pros and Cons of Ion-Torrent Next Generation Sequencing versus Terminal Restriction Fragment Length Polymorphism T-RFLP for Studying the Rumen Bacterial Community

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population.     

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.