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This study aimed to compare the microbiome profiles of patients with colorectal cancer (CRC, n  = 380) and colorectal adenomas (CRA, n  = 110) against generally healthy participants ( n  = 2,461) from various studies. The overarching objective was to conduct a real-life experiment and develop a robust machine learning model applicable to the general population. A total of 2,951 stool samples underwent a comprehensive analysis using the in-house MetaBakery pipeline. This included various data matrices such as microbial taxonomy, functional genes, enzymatic reactions, metabolic pathways, and predicted metabolites. The study found no statistically significant difference in microbial diversity among individuals. However, distinct clusters were identified for healthy, CRC, and CRA groups through linear discriminant analysis (LDA). Machine learning analysis demonstrated consistent model performance, indicating the potential of microbiome layers (microbial taxa, functional genes, enzymatic reactions, and metabolic pathways) as prediagnostic indicators for CRC and CRA. Notable biomarkers on the taxonomy level and microbial functionality (gene families, enzymatic reactions, and metabolic pathways) associated with CRC were identified. The research presents promising avenues for practical clinical applications, with potential validation on external clinical datasets in future studies., Competing Interests: LD was employed by the NU B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Murovec, Deutsch and Stres.)

An important aspect to an immersive experience in Virtual Reality is vection, defined as the illusion of self-motion. Much of the literature to date has explored strategies to maximize vection through manipulations of the visual stimulus (e.g., increasing speed) or the experimental context (e.g., framing of the study instructions). However, the role of individual differences (e.g., age, biological sex) in vection susceptibility has received little attention. The objective of the current study was to investigate the influence of individual-difference factors on vection perception in younger and older adults. Forty-six younger adults ( M age  = 25.1) and 39 older adults ( M age  = 72.4) completed assessments of personality traits, field dependence, and visual attention prior to observing a moving visual stimulus aimed at inducing circular vection. Vection was measured using self-reports of onset latency, duration, and intensity. Results indicated that, in both age groups, females experienced longer-lasting vection compared to males. Additionally, the level of field dependence was related to vection intensity and duration in males but not in females. Variability in vection intensity was best explained by a mixture of biological, perceptual, cognitive, and personality variables. Taken together, these findings suggest that individual factors are important for understanding differences in vection susceptibility., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

In this study, we present MetaBakery (http://metabakery.fe.uni-lj.si), an integrated application designed as a framework for synergistically executing the bioBakery workflow and associated utilities. MetaBakery streamlines the processing of any number of paired or unpaired fastq files, or a mixture of both, with optional compression (gzip, zip, bzip2, xz, or mixed) within a single run. MetaBakery uses programs such as KneadData (https://github.com/bioBakery/kneaddata), MetaPhlAn, HUMAnN and StrainPhlAn as well as integrated utilities and extends the original functionality of bioBakery. In particular, it includes MelonnPan for the prediction of metabolites and Mothur for calculation of microbial alpha diversity. Written in Python 3 and C++ the whole pipeline was encapsulated as Singularity container for efficient execution on various computing infrastructures, including large High-Performance Computing clusters. MetaBakery facilitates crash recovery, efficient re-execution upon parameter changes, and processing of large data sets through subset handling and is offered in three editions with bioBakery ingredients versions 4, 3 and 2 as versatile, transparent and well documented within the MetaBakery Users' Manual (http://metabakery.fe.uni-lj.si/metabakery_manual.pdf). It provides automatic handling of command line parameters, file formats and comprehensive hierarchical storage of output to simplify navigation and debugging. MetaBakery filters out potential human contamination and excludes samples with low read counts. It calculates estimates of alpha diversity and represents a comprehensive and augmented re-implementation of the bioBakery workflow. The robustness and flexibility of the system enables efficient exploration of changing parameters and input datasets, increasing its utility for microbiome analysis. Furthermore, we have shown that the MetaBakery tool can be used in modern biostatistical and machine learning approaches including large-scale microbiome studies., Competing Interests: LD was employed by The Nu B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Murovec, Deutsch, Osredkar and Stres.)

Vection is typically defined as the embodied illusion of self-motion in the absence of real physical movement through space. Vection can occur in real-life situations (e.g., 'train illusion') and in virtual environments and simulators. The vast majority of vection research focuses on vection caused by visual stimulation. Even though visually induced vection is arguably the most compelling type of vection, the role of nonvisual sensory inputs, such as auditory, biomechanical, tactile, and vestibular cues, have recently gained more attention. Non-visual cues can play an important role in inducing vection in two ways. First, nonvisual cues can affect the occurrence and strength of vection when added to corresponding visual information. Second, nonvisual cues can also elicit vection in the absence of visual information, for instance when observers are blindfolded or tested in darkness. The present paper provides a narrative review of the literature on multimodal contributions to vection. We will discuss both the theoretical and applied relevance of multisensory processing as related to the experience of vection and provide design considerations on how to enhance vection in various contexts.

Objective: Two studies were conducted to develop and validate a questionnaire to estimate individual susceptibility to visually induced motion sickness (VIMS)., Background: VIMS is a common side-effect when watching dynamic visual content from various sources, such as virtual reality, movie theaters, or smartphones. A reliable questionnaire predicting individual susceptibility to VIMS is currently missing. The aim was to fill this gap by introducing the Visually Induced Motion Sickness Susceptibility Questionnaire (VIMSSQ)., Methods: A survey and an experimental study were conducted. Survey: The VIMSSQ investigated the frequency of nausea, headache, dizziness, fatigue, and eyestrain when using different visual devices. Data were collected from a survey of 322 participants for the VIMSSQ and other related phenomena such as migraine. Experimental study: 23 participants were exposed to a VIMS-inducing visual stimulus. Participants filled out the VIMSSQ together with other questionnaires and rated their level of VIMS using the Simulator Sickness Questionnaire (SSQ)., Results: Survey: The most prominent symptom when using visual devices was eyestrain, and females reported more VIMS than males. A one-factor solution with good scale reliability was found for the VIMSSQ. Experimental study: Regression analyses suggested that the VIMSSQ can be useful in predicting VIMS ( R 2 = .34) as measured by the SSQ, particularly when combined with questions pertaining to the tendency to avoid visual displays and experience syncope ( R 2 = .59)., Conclusion: We generated normative data for the VIMSSQ and demonstrated its validity., Application: The VIMSSQ can become a valuable tool to estimate one's susceptibility to VIMS based on self-reports.

The illusion of self-motion (vection) is a multisensory phenomenon elicited by visual, auditory, tactile, or other sensory cues. Aging is often associated with changes in sensory acuity, visual motion perception, and multisensory integration, processes which may influence vection perception. However, age-related differences in vection have received little study to date. Thus, the objective of the present study was to investigate age-related differences in vection during multisensory stimulation. Nineteen younger adults and 19 older adults were exposed to rotating visual, auditory, and tactile stimuli (separately or in combination) at a speed of 45°/s inside a VR laboratory inducing circular vection. The size of the field-of-view (FOV) was large (240°), medium (75°), small (30°), or contained no visuals. Vection intensity and duration were reported verbally after each trial. Overall, older adults experienced significantly stronger and longer vection compared to younger adults. Additionally, there were main effects of FOV and sensory cues, such that larger FOVs and the presence of auditory and tactile stimulation increased vection ratings for both age groups. These findings support the idea that vection is a multisensory experience that can be elicited by visual, auditory, and tactile stimuli and demonstrates these effects for the first time in older adults.

Preterm birth (before 37 weeks gestation) accounts for ~10% of births worldwide and remains one of the leading causes of death in children under 5 years of age. Preterm born adults have been consistently shown to be at an increased risk for chronic disorders including cardiovascular, endocrine/metabolic, respiratory, renal, neurologic, and psychiatric disorders that result in increased death risk. Oxidative stress was shown to be an important risk factor for hypertension, metabolic syndrome and lung disease (reduced pulmonary function, long-term obstructive pulmonary disease, respiratory infections, and sleep disturbances). The aim of this study was to explore the differences between preterm and full-term male participants' levels of urine and fecal proton nuclear magnetic resonance ( 1 H-NMR) metabolomes, during rest and exercise in normoxia and hypoxia and to assess general differences in human gut-microbiomes through metagenomics at the level of taxonomy, diversity, functional genes, enzymatic reactions, metabolic pathways and predicted gut metabolites. Significant differences existed between the two groups based on the analysis of 1 H-NMR urine and fecal metabolomes and their respective metabolic pathways, enabling the elucidation of a complex set of microbiome related metabolic biomarkers, supporting the idea of distinct host-microbiome interactions between the two groups and enabling the efficient classification of samples; however, this could not be directed to specific taxonomic characteristics.

Physical inactivity is a worldwide health problem, an important risk for global mortality and is associated with chronic noncommunicable diseases. The aim of this study was to explore the differences in systemic urine 1 H-NMR metabolomes between physically active and inactive healthy young males enrolled in the X-Adapt project in response to controlled exercise (before and after the 3-day exercise testing and 10-day training protocol) in normoxic (21% O 2 ), normobaric (~1000 hPa) and normal-temperature (23 °C) conditions at 1 h of 50% maximal pedaling power output (W peak ) per day. Interrogation of the exercise database established from past X-Adapt results showed that significant multivariate differences existed in physiological traits between trained and untrained groups before and after training sessions and were mirrored in significant differences in urine pH, salinity, total dissolved solids and conductivity. Cholate, tartrate, cadaverine, lysine and N6-acetyllisine were the most important metabolites distinguishing trained and untrained groups. The relatively little effort of 1 h 50% W peak per day invested by the untrained effectively modified their resting urine metabolome into one indistinguishable from the trained group, which hence provides a good basis for the planning of future recommendations for health maintenance in adults, irrespective of the starting fitness value. Finally, the 3-day sessions of morning urine samples represent a good candidate biological matrix for future delineations of active and inactive lifestyles detecting differences unobservable by single-day sampling due to day-to-day variability.

A critical component to many immersive experiences in virtual reality (VR) is vection, defined as the illusion of self-motion. Traditionally, vection has been described as a visual phenomenon, but more recent research suggests that vection can be influenced by a variety of senses. The goal of the present study was to investigate the role of multisensory cues on vection by manipulating the availability of visual, auditory, and tactile stimuli in a VR setting. To achieve this, 24 adults (Mage = 25.04) were presented with a rotating stimulus aimed to induce circular vection. All participants completed trials that included a single sensory cue, a combination of two cues, or all three cues presented together. The size of the field of view (FOV) was manipulated across four levels (no-visuals, small, medium, full). Participants rated vection intensity and duration verbally after each trial. Results showed that all three sensory cues induced vection when presented in isolation, with visual cues eliciting the highest intensity and longest duration. The presence of auditory and tactile cues further increased vection intensity and duration compared to conditions where these cues were not presented. These findings support the idea that vection can be induced via multiple types of sensory inputs and can be intensified when multiple sensory inputs are combined.

General Unified Microbiome Profiling Pipeline (GUMPP) was developed for large scale, streamlined and reproducible analysis of bacterial 16S rRNA data and prediction of microbial metagenomes, enzymatic reactions and metabolic pathways from amplicon data. GUMPP workflow introduces reproducible data analyses at each of the three levels of resolution (genus; operational taxonomic units (OTUs); amplicon sequence variants (ASVs)). The ability to support reproducible analyses enables production of datasets that ultimately identify the biochemical pathways characteristic of disease pathology. These datasets coupled to biostatistics and mathematical approaches of machine learning can play a significant role in extraction of truly significant and meaningful information from a wide set of 16S rRNA datasets. The adoption of GUMPP in the gut-microbiota related research enables focusing on the generation of novel biomarkers that can lead to the development of mechanistic hypotheses applicable to the development of novel therapies in personalized medicine.

Microbial species play important roles in different environments and the production of high-quality genomes from metagenome data sets represents a major obstacle to understanding their ecological and evolutionary dynamics. Metagenome-Assembled Genomes Orchestra (MAGO) is a computational framework that integrates and simplifies metagenome assembly, binning, bin improvement, bin quality (completeness and contamination), bin annotation, and evolutionary placement of bins via detailed maximum-likelihood phylogeny based on multiple marker genes using different amino acid substitution models, next to average nucleotide identity analysis of genomes for delineation of species boundaries and operational taxonomic units. MAGO offers streamlined execution of the entire metagenomics pipeline, error checking, computational resource distribution and compatibility of data formats, governed by user-tailored pipeline processing. MAGO is an open-source-software package released in three different ways, as a singularity image and a Docker container for HPC purposes as well as for running MAGO on a commodity hardware, and a virtual machine for gaining a full access to MAGO underlying structure and source code. MAGO is open to suggestions for extensions and is amenable for use in both research and teaching of genomics and molecular evolution of genomes assembled from small single-cell projects or large-scale and complex environmental metagenomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In this study, nuclear magnetic resonance ( 1 H NMR) spectroscopic profiling was used to provide a more comprehensive view of microbial metabolites associated with poor reactor performance in a full-scale 4 MW mesophilic agricultural biogas plant under fully operational and also under inhibited conditions. Multivariate analyses were used to assess the significance of differences between reactors whereas artificial neural networks (ANN) were used to identify the key metabolites responsible for inhibition and their network of interaction. Based on the results of nm-MDS ordination the subsamples of each reactor were similar, but not identical, despite homogenization of the full-scale reactors before sampling. Hence, a certain extent of variability due to the size of the system under analysis was transferred into metabolome analysis. Multivariate analysis showed that fully active reactors were clustered separately from those containing inhibited reactor metabolites and were significantly different. Furthermore, the three distinct inhibited states were significantly different from each other. The inhibited metabolomes were enriched in acetate, caprylate, trimethylamine, thymine, pyruvate, alanine, xanthine and succinate. The differences in the metabolic fingerprint between inactive and fully active reactors observed in this study resembled closely the metabolites differentiating the (sub) acute rumen acidosis inflicted and healthy rumen metabolomes, creating thus favorable conditions for the growth and activity of pathogenic bacteria. The consistency of our data with those reported before for rumen ecosystems shows that 1 H NMR based metabolomics is a reliable approach for the evaluation of metabolic events at full-scale biogas reactors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We explored the metagenomic, metabolomic and trace metal makeup of intestinal microbiota and environment in healthy male participants during the run-in (5 day) and the following three 21-day interventions: normoxic bedrest (NBR), hypoxic bedrest (HBR) and hypoxic ambulation (HAmb) which were carried out within a controlled laboratory environment (circadian rhythm, fluid and dietary intakes, microbial bioburden, oxygen level, exercise). The fraction of inspired O 2 (F i O 2 ) and partial pressure of inspired O 2 (P i O 2 ) were 0.209 and 133.1 ± 0.3 mmHg for the NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg (~4,000 m simulated altitude) for HBR and HAmb interventions, respectively. Shotgun metagenomes were analyzed at various taxonomic and functional levels, 1 H- and 13 C -metabolomes were processed using standard quantitative and human expert approaches, whereas metals were assessed using X-ray fluorescence spectrometry. Inactivity and hypoxia resulted in a significant increase in the genus Bacteroides in HBR, in genes coding for proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence, defense and mucin degradation, such as beta-galactosidase (EC3.2.1.23), α-L-fucosidase (EC3.2.1.51), Sialidase (EC3.2.1.18), and α-N-acetylglucosaminidase (EC3.2.1.50). In contrast, the microbial metabolomes, intestinal element and metal profiles, the diversity of bacterial, archaeal and fungal microbial communities were not significantly affected. The observed progressive decrease in defecation frequency and concomitant increase in the electrical conductivity (EC) preceded or took place in absence of significant changes at the taxonomic, functional gene, metabolome and intestinal metal profile levels. The fact that the genus Bacteroides and proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence and mucin degradation were enriched at the end of HBR suggest that both constipation and EC decreased intestinal metal availability leading to modified expression of co-regulated genes in Bacteroides genomes. Bayesian network analysis was used to derive the first hierarchical model of initial inactivity mediated deconditioning steps over time. The PlanHab wash-out period corresponded to a profound life-style change (i.e., reintroduction of exercise) that resulted in stepwise amelioration of the negative physiological symptoms, indicating that exercise apparently prevented the crosstalk between the microbial physiology, mucin degradation and proinflammatory immune activities in the host.

We explored the assembly of intestinal microbiota in healthy male participants during the randomized crossover design of run-in (5 day) and experimental phases (21-day normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, with balanced fluid and dietary intakes, controlled circadian rhythm, microbial ambiental burden and 24/7 medical surveillance. The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) were 0.209 and 133.1 ± 0.3 mmHg for NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg for both hypoxic variants (HBR and HAmb; ~4000 m simulated altitude), respectively. A number of parameters linked to intestinal environment such as defecation frequency, intestinal electrical conductivity (IEC), sterol and polyphenol content and diversity, indole, aromaticity and spectral characteristics of dissolved organic matter (DOM) were measured (64 variables). The structure and diversity of bacterial microbial community was assessed using 16S rRNA amplicon sequencing. Inactivity negatively affected frequency of defecation and in combination with hypoxia increased IEC (p < 0.05). In contrast, sterol and polyphenol diversity and content, various characteristics of DOM and aromatic compounds, the structure and diversity of bacterial microbial community were not significantly affected over time. A new in-house PlanHab database was established to integrate all measured variables on host physiology, diet, experiment, immune and metabolic markers (n = 231). The observed progressive decrease in defecation frequency and concomitant increase in IEC suggested that the transition from healthy physiological state towards the developed symptoms of low magnitude obesity-related syndromes was dose dependent on the extent of time spent in inactivity and preceded or took place in absence of significant rearrangements in bacterial microbial community. Species B. thetaiotamicron, B. fragilis, B. dorei and other Bacteroides with reported relevance for dysbiotic medical conditions were significantly enriched in HBR, characterized with most severe inflammation symptoms, indicating a shift towards host mucin degradation and proinflammatory immune crosstalk.

We explored the assembly of intestinal microbiota in healthy male participants during the run-in (5 day) and experimental phases [21-day normoxic bed rest (NBR), hypoxic bedrest (HBR)], and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, balanced fluid, and dietary intakes, controlled circadian rhythm, microbial ambiental burden, and 24/7 medical surveillance. The fraction of inspired O 2 (F i O 2 ) and partial pressure of inspired O 2 (P i O 2 ) were 0.209 and 133.1 ± 0.3 mmHg for NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg for both hypoxic variants (HBR and HAmb; ~4,000 m simulated altitude), respectively. A number of parameters linked to intestinal transit spanning Bristol Stool Scale, defecation rates, zonulin, α 1 -antitrypsin, eosinophil derived neurotoxin, bile acids, reducing sugars, short chain fatty acids, total soluble organic carbon, water content, diet composition, and food intake were measured (167 variables). The abundance, structure, and diversity of butyrate producing microbial community were assessed using the two primary bacterial butyrate synthesis pathways, butyryl-CoA: acetate CoA-transferase ( but ) and butyrate kinase ( buk ) genes. Inactivity negatively affected fecal consistency and in combination with hypoxia aggravated the state of gut inflammation ( p < 0.05). In contrast, gut permeability, various metabolic markers, the structure, diversity, and abundance of butyrate producing microbial community were not significantly affected. Rearrangements in the butyrate producing microbial community structure were explained by experimental setup (13.4%), experimentally structured metabolites (12.8%), and gut metabolite-immunological markers (11.9%), with 61.9% remaining unexplained. Many of the measured parameters were found to be correlated and were hence omitted from further analyses. The observed progressive increase in two immunological intestinal markers suggested that the transition from healthy physiological state toward the developed symptoms of low magnitude obesity-related syndromes was primarily driven by the onset of inactivity (lack of exercise in NBR) that were exacerbated by systemic hypoxia (HBR) and significantly alleviated by exercise, despite hypoxia (HAmb). Butyrate producing community in colon exhibited apparent resilience toward short-term modifications in host exercise or hypoxia. Progressive constipation (decreased intestinal motility) and increased local inflammation marker suggest that changes in microbial colonization and metabolism were taking place at the location of small intestine.

The aim of this study was to develop and validate a community supported online infrastructure and bioresource for methane yield data and accompanying metadata collected from published literature. In total, 1164 entries described by 15,749 data points were assembled. Analysis of data collection showed little congruence in reporting of methodological approaches. The largest identifiable source of variation in reported methane yields was represented by authorship (i.e. substrate batches within particular substrate class) within which experimental scales (volumes (0.02-5l), incubation temperature (34-40 °C) and % VS of substrate played an important role (p < 0.05, npermutations = 999) as well. The largest fraction of variability, however, remained unaccounted for and thus unexplained (> 63%). This calls for reconsideration of accepted approaches to reporting data in currently published literature to increase capacity to service industrial decision making to a greater extent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The Himalaya with its altitude and geographical position forms a barrier to atmospheric transport, which produces much aqueous-particle monsoon precipitation and makes it the largest continuous ice-covered area outside polar regions. There is a paucity of data on high-altitude microbial communities, their native environments and responses to environmental-spatial variables relative to seasonal and deglaciation events., Methodology/principal Findings: Soils were sampled along altitude transects from 5000 m to 6000 m to determine environmental, spatial and seasonal factors structuring bacterial communities characterized by 16 S rRNA gene deep sequencing. Dust traps and fresh-snow samples were used to assess dust abundance and viability, community structure and abundance of dust associated microbial communities. Significantly different habitats among the altitude-transect samples corresponded to both phylogenetically distant and closely-related communities at distances as short as 50 m showing high community spatial divergence. High within-group variability that was related to an order of magnitude higher dust deposition obscured seasonal and temporal rearrangements in microbial communities. Although dust particle and associated cell deposition rates were highly correlated, seasonal dust communities of bacteria were distinct and differed significantly from recipient soil communities. Analysis of closest relatives to dust OTUs, HYSPLIT back-calculation of airmass trajectories and small dust particle size (4-12 µm) suggested that the deposited dust and microbes came from distant continental, lacustrine and marine sources, e.g. Sahara, India, Caspian Sea and Tibetan plateau. Cyanobacteria represented less than 0.5% of microbial communities suggesting that the microbial communities benefitted from (co)deposited carbon which was reflected in the psychrotolerant nature of dust-particle associated bacteria., Conclusions/significance: The spatial, environmental and temporal complexity of the high-altitude soils of the Himalaya generates ongoing disturbance and colonization events that subject heterogeneous microniches to stochastic colonization by far away dust associated microbes and result in the observed spatially divergent bacterial communities.

The exponential growth of available DNA sequences and the increased interoperability of biological information is triggering intergovernmental efforts aimed at increasing the access, dissemination, and analysis of sequence data. Achieving the efficient storage and processing of DNA material is an important goal that parallels well with the foreseen coding standardization on the horizon. This paper proposes novel coding approaches, for both the dissemination and processing of sequences, where the speed of the DNA processing is shown to be boosted by exploring more than the normally utilized eight bits for encoding a single nucleotide. Further gains are achieved by encoding the nucleotides together with their trailing alignment information as a single 64-bit data structure. The paper also proposes a slight modification to the established FASTA scheme in order to improve on its representation of alignment information. The significance of the propositions is confirmed by the encouraging results from empirical tests., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Summary: BEsTRF (Best Estimated T-RF) provides a standalone environment for analyzing primers-enzymes-gene section combinations used in terminal-restriction fragment length polymorphism (T-RFLP) for its optimal resolution. User-defined sequence databases of several hundred thousand DNA sequences can be explored and the resolution of user-specified sets of primers and restriction endonucleases can be analyzed on either forward or reverse terminal fragments. Sequence quality, primer mismatches, insertions and deletions can be controlled and each primer pair-specific sequence collections can be exported for downstream analyses. The configuration for a novel T-RFLP population profiling using rpoB gene (DNA-directed RNA polymerase, beta subunit) on forward fluorescently labeled primer are presented., Availability: BEsTRF is freely available at http://lie.fe.uni-lj.si/bestrf and can be downloaded from the same site. The online protocol, numerous primer and enzyme dictionaries, sequence collections and results generated during this work for various genes are available at our website http://lie.fe.uni-lj.si/bestrf.