Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota

Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.     

Detection of Phanerochaete chrysosporium in soil by PCR and restriction enzyme analysis.

A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA. Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 micrograms of total DNA extracted from 1 g of soil.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

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

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

Direct Species Identification of Common Pathogenic Dermatophyte Fungi in Clinical Specimens by Semi-nested PCR and Restriction Fragment Length Polymorphism

OBJECTIVE: To seek a rapid and reliable molecular biology method to identify the common pathogenic dermatophyte fungi from clinical samples. METHOD: The genome DNA was extracted from cultured strains of seven common dermatophyte fungi species and part of each positive clinical specimen by microscopy. Intergenic spacer regions of ribosomal DNA (ITS) were amplified by semi-nested PCR (snPCR) with three universal primers (NS5, ITS1, and ITS4) for fungi. The amplified products were digested with two restriction endonucleases (BciT130 I, Dde I), the Restriction Fragment Length Polymorphism(RFLP). The rest of each clinical specimen was cultured in Sabouraud's Agar medium. Then the results of RFLP were compared with the traditional culture results. RESULTS: The digestion of seven common dermatophyte fungi produced seven different restriction profiles. Restriction profiles of 17 clinical specimens matched, respectively, to that of the cultured strains, and 14 profiles of the 17 ones matched the culture result completely. The coincidence was 100.0%. CONCLUSIONS: snPCR-RFLP analysis of intergenic spacer regions of ribosomal DNA is a valuable method of exactness and clarity for species identification of common dermatophyte fungi from clinical specimens.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.     

ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.     

A diverse population of introns in the nuclear ribosomal genes of ericoid mycorrhizal fungi includes elements with sequence similarity to endonuclease-coding genes

Ericoid mycorrhizal fungi form symbioses with the roots of members of the Ericales. Although only two genera have been identified in culture, the taxonomic diversity of ericoid symbionts is certainly wider. Genetic variation among 40 ericoid fungal isolates was investigated in this study. PCR amplification of the nuclear small-subunit ribosomal DNA (SSU rDNA) and of the internal transcribed spacer (ITS), followed by sequencing, led to the discovery of DNA insertions of various sizes in the SSU rDNA of most isolates. They reached sizes of almost 1,800 bp and occurred in up to five different insertion sites. Their positions and sizes were generally correlated with morphological and ITS-RFLP grouping of the isolates, although some insertions were found to be optional among isolates of the same species, and insertions were not always present in all SSU rDNA repeats within an isolate. Most insertions were identified as typical group I introns, possessing the conserved motifs characteristic of this group. However, other insertions lack these motifs and form a distinct group that includes other fungal ribosomal introns. Alignments with almost 70 additional sequences from fungal nuclear SSU rDNA introns indicate that introns inserted at the same site along the rDNA gene are generally homologous, but they also suggest the possibility of some horizontal transfers. Two of the ericoid fungal introns showed strong homology with a conserved motif found in endonuclease genes from nuclear rDNA introns.     

Soil chemical properties,rather than landscape context,influence woodland fungal communities along an urban‐rural gradient

With the expansion of cities around the world there is a growing interest in the factors that influence biodiversity and ecosystem processes in urban areas. Fungi are exceptionally diverse and play key roles in ecosystem function, yet despite predictions of negative impacts due to urbanization, fungi have been generally overlooked in urban ecological studies. We surveyed fungi in 16 remnant river red gum (Eucalyptus camaldulensis: Myrtaceae) woodlands along a gradient of 4–35 km from the city of Melbourne (south‐east Australia). Using both sporocarp surveys and terminal restriction fragment length polymorphism (T‐RFLP; primer pair ITS1‐F‐ITS4), we examined relationships between fungal community composition, landscape context (i.e. urbanization) and soil physicochemical properties. Community compositions from sporocarp data were significantly correlated with those from T‐RFLP data, largely because of correlations with ectomycorrhizal sporocarps (Spearman rank correlation coefficients ρ 0.31–0.42) rather than saprotrophic fungi (ρ 0.18–0.21). Principal components analysis of soil properties and non‐metric multidimensional scaling ordinations of fungal community composition showed no clear separation of sites according to urbanization, and there were no significant correlations between fungal community composition and urbanization. However, fungal community composition was significantly correlated with soil chemical properties (ρ 0.41–0.55). These data suggest that site‐scale soil properties, and associated effects of past and current land management activities, were more important in determining fungal community composition than the landscape‐level influences of urbanization.     

Molecular diversity of soil basidiomycete communities in northern-central New South Wales, Australia

Basidiomycete communities were profiled using terminal RFLP (TRFLP) and amplified ribosomal DNA restriction analysis (ARDRA) approaches at seven field sites under differing land use in northern-central New South Wales (NSW), Australia. TRFLP data indicated greater basidiomycete species richness at sites with natural vegetation. Sixty-seven basidiomycete ARDRA-types were detected. Various putatively ectomycorrhizal fungi were detected at all sites with native vegetation. Most ectomycorrhizal taxa had affinities to the genus Tomentella, while two Pisolithus taxa and putatively ectomycorrhizal Cantharellales taxa were also detected. Although soils under woodland or grassland communities supported a range of putatively saprotrophic taxa, only members of the Ceratobasidiales were detected in soils under agricultural land use. This study is the first investigation of fungal communities in soils of northern-central NSW, Australia.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

Soil Bacterial and Fungal Community Structure Across a Range of Unimproved and Semi-Improved Upland Grasslands

Changes in soil microbial community structure due to improvement are often attributed to concurrent shifts in floristic community composition. The bacterial and fungal communities of unimproved and semi-improved (as determined by floristic classification) grassland soils were studied at five upland sites on similar geological substrata using both broad-scale (microbial activity and fungal biomass) and molecular [terminal restriction fragment length polymorphism (TRFLP), automated ribosomal intergenic spacer analysis (ARISA)] approaches. It was hypothesized that microbial community structure would be similar in soils from the same grassland type, and that grassland vegetation classifications could thus be used as predictors of microbial community structure. Microbial community measurements varied widely according to both site and grassland type, and trends in the effect of grassland improvement differed between sites. These results were consistent with those from similar studies, and indicated that floristic community composition was not a stable predictor of microbial community structure across sites. This may indicate a lack of correlation between grassland plant composition and soil microbial community structure, or that differences in soil chemistry between sites had larger impacts on soil microbial populations than plant-related effects.     

Exploring the accuracy of amplicon‐based internal transcribed spacer markers for a fungal community

With the continual improvement in high‐throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon‐based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both primer selection and the experimental procedure require meticulous verification. Here, we computationally and experimentally evaluated the accuracy and specificity of three widely used or newly designed internal transcribed spacer (ITS) primer sets (ITS1F/ITS2, gITS7/ITS4 and 5.8S‐Fun/ITS4‐Fun). In silico evaluation revealed that primer coverage varied at different taxonomic levels due to differences in degeneracy and the location of primer sets. Using even and staggered mock community standards, we identified different proportions of chimeric and mismatch reads generated by different primer sets, as well as great variation in species abundances, suggesting that primer selection would affect the results of amplicon‐based metabarcoding studies. Choosing proofreading and high‐fidelity polymerase (KAPA HiFi) could significantly reduce the percentage of chimeric and mismatch sequences, further reducing inflation of operational taxonomic units. Moreover, for two types of environmental fungal communities, plant endophytic and soil fungi, it was demonstrated that the three primer sets could not reach a consensus on fungal community composition or diversity, and that primer selection, not experimental treatment, determines observed soil fungal community diversity and composition. Future DNA marker surveys should pay greater attention to potential primer effects and improve the experimental scheme to increase credibility and accuracy.     

What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities

Arbuscular mycorrhizal (AM) fungi are widely distributed microbes that form obligate symbioses with the majority of terrestrial plants, altering nutrient transfers between soils and plants, thereby profoundly affecting plant growth and ecosystem properties. Molecular methods are commonly used in the study of AM fungal communities. However, the biases associated with PCR amplification of these organisms and their ability to be utilized quantitatively has never been fully tested. We used Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to characterise artificial community templates containing known quantities of defined AM fungal genotypes. This was compared to a parallel in silico analysis that predicted the results of this experiment in the absence of bias. The data suggest that when used quantitatively the TRFLP protocol tested is a powerful, repeatable method for AM fungal community analysis. However, we suggest some limitations to its use for population-level analyses. We found no evidence of PCR bias, supporting the quantitative use of other PCR-based methods for the study of AM fungi such as next generation amplicon sequencing. This finding greatly improves our confidence in methods that quantitatively examine AM fungal communities, providing a greater understanding of the ecology of these important fungi.     

ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints

ribosort is a computer package for convenient editing of automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) data. It is designed to eliminate the labourious task of manually classifying community fingerprints in microbial ecology studies. This program automatically assigns detected fragments and their respective relative abundances to appropriate ribotypes. It permits simultaneous sorting of multiple profiles and facilitates direct workflow from TRFLP and ARISA output through to community analyses. ribosort also provides several options to merge repeat profiles of a sample into a single composite profile. By creating a 'ribotypes by samples' matrix ready for statistical analyses, use of the package saves time and simplifies the preparation of DNA fingerprint data sets for statistical analysis. In addition, ribosort performs exploratory analysis on the data by creating multidimensional scaling plots that compare the similarity of sample profiles using the statistical software r.