Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.     

Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.     

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol–chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.     

Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities

DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities.     

Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes

Shotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias against Verrucomicrobia in a commonly used V6-V8 primer set, as well as discovering likely bias against Actinobacteria and for Verrucomicrobia in a commonly used V4 primer set. This pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure. The pipeline can scale to handle large amounts of soil metagenomic data (5 Gb memory and 5 central processing unit hours to process 38 Gb [1 lane] of trimmed Illumina HiSeq2500 data) and is freely available at https://github.com/dib-lab/SSUsearch under a BSD license.     

Barcoded primers used in multiplex amplicon pyrosequencing bias amplification

"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

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.     

Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.     

Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions of the templates. This bias was strongly dependent on the number of cycles of replication. The results fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids.     

Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products

Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions. For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests. During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates. We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days. The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference. The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively. The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration. The effects on Bacteria, Archaea, and Pseudomonas were investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting. Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress. In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities. We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated.     

Community structure of Archaea and Bacteria in a profundal lake sediment Lake Kinneret (Israel)

The microbial community structure of an anoxic profundal lake sediment, i.e., subtropical Lake Kinneret, was analysed with respect to its composition by culture-independent molecular methods including terminal restriction fragment length polymorphism (T-RFLP) analysis, comparative sequence analysis, and quantitative real-time PCR. In particular we were interested in the structure, species composition, and relative abundance of the overall microbial community in the methanogenic sediment layer (0-10 cm depth). Pairwise comparison of archaeal and bacterial 16S rRNA gene T-RFLP profiles obtained from three independent samplings indicated stability of the microbial community. The numbers of Archaea and Bacteria, quantified by real-time PCR, amounted to about 10(8) and 10(10) 16S rRNA gene copies cm(-3) sediment, respectively, suggesting that Archaea may account for only a minor fraction (approximately 1%) of the total prokaryotic community. Hydrogenotrophic Methanomicrobiales and acetoclastic Methanosaeta spp. dominated T-RFLP profiles of the archaeal community. T-RFLP profiles of the bacterial community were dominated by Deltaproteobacteria, sulphate reducers and syntrophs in particular. The second most abundant group was assigned to the Bacteroidetes-Chlorobi-group. Only one bacterial group, which was affiliated with halorespiring bacteria of subphylum II of the Chloroflexi, showed variation in abundance within the sediment samples investigated. Our study gives a comprehensive insight into the structure of the bacterial and archaeal community of a profundal lake sediment, indicating that sulphate reducers, syntrophs, bacteroidetes, halorespirers and methanogens are of particular importance in Lake Kinneret sediment.     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).     

Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots

Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.     

Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure

The aim of this study was to assess the effect of sample pooling on the portrayal of ciliate community structure and composition in intertidal sediment samples. Molecular ciliate community profiles were obtained from nine biological replicates distributed in three discrete sampling plots and from samples that were pooled prior to RNA extraction using terminal restriction fragment polymorphism (T-RFLP) analyses of SSU rRNA. Comparing the individual replicates of one sampling plot with each other, we found a differential variability among the individual biological replicates. T-RFLP profiles of pooled samples displayed a significantly different community composition compared with the cumulative individual biological replicate samples. We conclude that sample pooling obscures diversity patterns in ciliate and possibly also other microbial eukaryote studies. However, differences between pooled samples and replicates were less pronounced when community structure was analyzed. We found that the most abundant T-RFLP peaks were generally shared between biological replicates and pooled samples. Assuming that the most abundant taxa in an ecosystem under study are also the ones driving ecosystem processes, sample pooling may still be effective for the analyses of ecological key players.