Tools for T-RFLP data analysis using Excel

Background

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed.

Results

The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data.

Conclusions

The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0361-7) contains supplementary material, which is available to authorized users.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

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.     

Modified T-RFLP methods for taxonomic interpretation of T-RF

Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.     

Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.Over the last 2 decades, the development of molecular biology tools has led to the emergence of a new discipline, molecular microbial ecology. The overall structural diversity of microbial communities can be examined easily using PCR-based strategies (6), usually targeting the 16S rRNA gene as a universal genetic marker of prokaryotes. Genotyping approaches avoid current limitations of cultivation methods, which only poorly reflect the phylogenetic diversity of microbial communities (12). The principles, technical aspects, and limitations of commonly employed methods were recently reviewed (10). Among these methods, terminal restriction fragment length polymorphism (T-RFLP) has proved to be invaluable for rapid characterization of the composition and dynamics of species-rich samples (13). Compared to other approaches, T-RFLP is semiquantitative and combines high levels of sensitivity, resolution, and reproducibility (see Table S1 in the supplemental material). Taxonomic diversity of microbial communities is evaluated by using the strain-dependent variability of restriction sites within a conserved PCR-amplified DNA fragment. The terminal restriction fragments (T-RFs) of digested PCR products appear as chromatographic peaks after size-dependent electrophoretic separation due to a fluorescent tag attached to one of the primers used for PCR. The relative abundance of peaks is evaluated, and fragment lengths are estimated using a fluorescent internal size standard comigrating with the sample (5). The estimated lengths corresponding to the T-RFLP peaks obtained are compared to databases of T-RF sizes generated by in silico digestion of known 16S rRNA gene sequences with commonly used restriction enzymes for phylogenetic assignment (13). However, estimation of T-RF lengths from experimental chromatograms is biased by the fact that differences in the electrophoretic properties of the two different fluorescent dyes used to distinguish sample fragments from the size marker significantly affect fragment migration (7, 11). Discrepancies greater than 6 nucleotides (nt), depending on the length of the fragment, have been reported between expected and experimentally estimated fragment lengths (7). This causes errors in phylogenetic assignments and may in turn lead to erroneous inferences regarding the functional aspects of the microbial communities under investigation. Another drawback of T-RFLP is the difficulty of retrieving sequence information directly from experimental T-RFs, since additional construction of representative 16S rRNA gene libraries is required to obtain such information.Here we propose an experimental strategy to circumvent current limitations of T-RFLP and facilitate characterization of microbial communities. First, we propose an optimized protocol for T-RFLP that yields reliable T-RF sizes. Second, we describe use of denaturing high-performance liquid chromatography (D-HPLC) as an alternative to cloning in order to gain direct access to DNA sequence information. D-HPLC, an emerging technique for microbial community profiling (1, 4), enables collection of DNA fragments separated on the basis of differences in sequence, sequence length, and G+C content at a partially denaturing temperature. The unambiguous phylogenetic characterization of a model microbial assembly of five reference strains is described as proof of principle of the usefulness of the proposed strategy.     

Systematic exploration of guide-tree topology effects for small protein alignments

Background

Guide-trees are used as part of an essential heuristic to enable the calculation of multiple sequence alignments. They have been the focus of much method development but there has been little effort at determining systematically, which guide-trees, if any, give the best alignments. Some guide-tree construction schemes are based on pair-wise distances amongst unaligned sequences. Others try to emulate an underlying evolutionary tree and involve various iteration methods.

Results

We explore all possible guide-trees for a set of protein alignments of up to eight sequences. We find that pairwise distance based default guide-trees sometimes outperform evolutionary guide-trees, as measured by structure derived reference alignments. However, default guide-trees fall way short of the optimum attainable scores. On average chained guide-trees perform better than balanced ones but are not better than default guide-trees for small alignments.

Conclusions

Alignment methods that use Consistency or hidden Markov models to make alignments are less susceptible to sub-optimal guide-trees than simpler methods, that basically use conventional sequence alignment between profiles. The latter appear to be affected positively by evolutionary based guide-trees for difficult alignments and negatively for easy alignments. One phylogeny aware alignment program can strongly discriminate between good and bad guide-trees. The results for randomly chained guide-trees improve with the number of sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-338) contains supplementary material, which is available to authorized users.     

Ammonia oxidizing bacteria community dynamics in a pilot-scale wastewater treatment plant

Background

Chemoautotrophic ammonia oxidizing bacteria (AOB) have the metabolic ability to oxidize ammonia to nitrite aerobically. This metabolic feature has been widely used, in combination with denitrification, to remove nitrogen from wastewater in wastewater treatment plants (WWTPs). However, the relative influence of specific deterministic environmental factors to AOB community dynamics in WWTP is uncertain. The ecological principles underlying AOB community dynamics and nitrification stability and how they are related are also poorly understood.

Methodology/Principal Findings

The community dynamics of ammonia oxidizing bacteria (AOB) in a pilot-scale WWTP were monitored over a one-year period by Terminal Restriction Fragment Length Polymorphism (T-RFLP). During the study period, the effluent ammonia concentrations were almost below 2 mg/L, except for the first 60 days, indicting stable nitrification. T-RFLP results showed that, during the test period with stable nitrification, the AOB community structures were not stable, and the average change rate (every 15 days) of AOB community structures was 10%±8%. The correlations between T-RFLP profiles and 10 operational and environmental parameters were tested by Canonical Correlation Analysis (CCA) and Mantel test. The results indicated that the dynamics of AOB community correlated most strongly with Dissolved Oxygen (DO), effluent ammonia, effluent Biochemical Oxygen Demand (BOD) and temperature.

Conclusions/Significance

This study suggests that nitrification stability is not necessarily accompanied by a stable AOB community, and provides insight into parameters controlling the AOB community dynamics within bioreactors with stable nitrification.     

Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array

Background

A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA).

Methodology/Principal Findings

The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment.

Conclusions

Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.     

Enhancing HMM-based protein profile-profile alignment with structural features and evolutionary coupling information

Background

Protein sequence profile-profile alignment is an important approach to recognizing remote homologs and generating accurate pairwise alignments. It plays an important role in protein sequence database search, protein structure prediction, protein function prediction, and phylogenetic analysis.

Results

In this work, we integrate predicted solvent accessibility, torsion angles and evolutionary residue coupling information with the pairwise Hidden Markov Model (HMM) based profile alignment method to improve profile-profile alignments. The evaluation results demonstrate that adding predicted relative solvent accessibility and torsion angle information improves the accuracy of profile-profile alignments. The evolutionary residue coupling information is helpful in some cases, but its contribution to the improvement is not consistent.

Conclusion

Incorporating the new structural information such as predicted solvent accessibility and torsion angles into the profile-profile alignment is a useful way to improve pairwise profile-profile alignment methods.     

Topological characterization of neuronal arbor morphology via sequence representation: II - global alignment

Background

The increasing abundance of neuromorphological data provides both the opportunity and the challenge to compare massive numbers of neurons from a wide diversity of sources efficiently and effectively. We implemented a modified global alignment algorithm representing axonal and dendritic bifurcations as strings of characters. Sequence alignment quantifies neuronal similarity by identifying branch-level correspondences between trees.

Results

The space generated from pairwise similarities is capable of classifying neuronal arbor types as well as, or better than, traditional topological metrics. Unsupervised cluster analysis produces groups that significantly correspond with known cell classes for axons, dendrites, and pyramidal apical dendrites. Furthermore, the distinguishing consensus topology generated by multiple sequence alignment of a group of neurons reveals their shared branching blueprint. Interestingly, the axons of dendritic-targeting interneurons in the rodent cortex associates with pyramidal axons but apart from the (more topologically symmetric) axons of perisomatic-targeting interneurons.

Conclusions

Global pairwise and multiple sequence alignment of neurite topologies enables detailed comparison of neurites and identification of conserved topological features in alignment-defined clusters. The methods presented also provide a framework for incorporation of additional branch-level morphological features. Moreover, comparison of multiple alignment with motif analysis shows that the two techniques provide complementary information respectively revealing global and local features.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0605-1) contains supplementary material, which is available to authorized users.     

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.     

Fluorescent protein-based methods for on-plate screening of gene insertion

Background

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.

Methodology/Principal Findings

Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.

Conclusions/Significance

The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening.     

ReformAlign: improved multiple sequence alignments using a profile-based meta-alignment approach

Background

Obtaining an accurate sequence alignment is fundamental for consistently analyzing biological data. Although this problem may be efficiently solved when only two sequences are considered, the exact inference of the optimal alignment easily gets computationally intractable for the multiple sequence alignment case. To cope with the high computational expenses, approximate heuristic methods have been proposed that address the problem indirectly by progressively aligning the sequences in pairs according to their relatedness. These methods however are not flexible to change the alignment of an already aligned group of sequences in the view of new data, resulting thus in compromises on the quality of the deriving alignment. In this paper we present ReformAlign, a novel meta-alignment approach that may significantly improve on the quality of the deriving alignments from popular aligners. We call ReformAlign a meta-aligner as it requires an initial alignment, for which a variety of alignment programs can be used. The main idea behind ReformAlign is quite straightforward: at first, an existing alignment is used to construct a standard profile which summarizes the initial alignment and then all sequences are individually re-aligned against the formed profile. From each sequence-profile comparison, the alignment of each sequence against the profile is recorded and the final alignment is indirectly inferred by merging all the individual sub-alignments into a unified set. The employment of ReformAlign may often result in alignments which are significantly more accurate than the starting alignments.

Results

We evaluated the effect of ReformAlign on the generated alignments from ten leading alignment methods using real data of variable size and sequence identity. The experimental results suggest that the proposed meta-aligner approach may often lead to statistically significant more accurate alignments. Furthermore, we show that ReformAlign results in more substantial improvement in cases where the starting alignment is of relatively inferior quality or when the input sequences are harder to align.

Conclusions

The proposed profile-based meta-alignment approach seems to be a promising and computationally efficient method that can be combined with practically all popular alignment methods and may lead to significant improvements in the generated alignments.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-265) contains supplementary material, which is available to authorized users.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Epitopemap: a web application for integrated whole proteome epitope prediction

Background

Predictions of MHC binding affinity are commonly used in immunoinformatics for T cell epitope prediction. There are multiple available methods, some of which provide web access. However there is currently no convenient way to access the results from multiple methods at the same time or to execute predictions for an entire proteome at once.

Results

We designed a web application that allows integration of multiple epitope prediction methods for any number of proteins in a genome. The tool is a front-end for various freely available methods. Features include visualisation of results from multiple predictors within proteins in one plot, genome-wide analysis and estimates of epitope conservation.

Conclusions

We present a self contained web application, Epitopemap, for calculating and viewing epitope predictions with multiple methods. The tool is easy to use and will assist in computational screening of viral or bacterial genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0659-0) contains supplementary material, which is available to authorized users.     

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.     

YOC,A new strategy for pairwise alignment of collinear genomes

Background

Comparing and aligning genomes is a key step in analyzing closely related genomes. Despite the development of many genome aligners in the last 15 years, the problem is not yet fully resolved, even when aligning closely related bacterial genomes of the same species. In addition, no procedures are available to assess the quality of genome alignments or to compare genome aligners.

Results

We designed an original method for pairwise genome alignment, named YOC, which employs a highly sensitive similarity detection method together with a recent collinear chaining strategy that allows overlaps. YOC improves the reliability of collinear genome alignments, while preserving or even improving sensitivity. We also propose an original qualitative evaluation criterion for measuring the relevance of genome alignments. We used this criterion to compare and benchmark YOC with five recent genome aligners on large bacterial genome datasets, and showed it is suitable for identifying the specificities and the potential flaws of their underlying strategies.

Conclusions

The YOC prototype is available at https://github.com/ruricaru/YOC. It has several advantages over existing genome aligners: (1) it is based on a simplified two phase alignment strategy, (2) it is easy to parameterize, (3) it produces reliable genome alignments, which are easier to analyze and to use.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0530-3) contains supplementary material, which is available to authorized users.