Erik Kristiansson

Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification

The internal transcribed spacer (ITS) region of the nuclear ribosomal repeat unit is the most popular locus for species identification and subgeneric phylogenetic inference in sequence-based mycological research. The region is known to show certain variability even within species, although its intraspecific variability is often held to be limited and clearly separated from interspecific variability. The existence of such a divide between intra- and interspecific variability is implicitly assumed by automated approaches to species identification, but whether intraspecific variability indeed is negligible within the fungal kingdom remains contentious. The present study estimates the intraspecific ITS variability in all fungi presently available to the mycological community through the international sequence databases. Substantial differences were found within the kingdom, and the results are not easily correlated to the taxonomic affiliation or nutritional mode of the taxa considered. No single unifying yet stringent upper limit for intraspecific variability, such as the canonical 3% threshold, appears to be applicable with the desired outcome throughout the fungi. Our results caution against simplified approaches to automated ITS-based species delimitation and reiterate the need for taxonomic expertise in the translation of sequence data into species names.

Taxonomic Reliability of DNA Sequences in Public Sequence Databases: A Fungal Perspective

Background DNA sequences are increasingly seen as one of the primary information sources for species identification in many organism groups. Such approaches, popularly known as barcoding, are underpinned by the assumption that the reference databases used for comparison are sufficiently complete and feature correctly and informatively annotated entries. Methodology/Principal Findings The present study uses a large set of fungal DNA sequences from the inclusive International Nucleotide Sequence Database to show that the taxon sampling of fungi is far from complete, that about 20% of the entries may be incorrectly identified to species level, and that the majority of entries lack descriptive and up-to-date annotations. Conclusions The problems with taxonomic reliability and insufficient annotations in public DNA repositories form a tangible obstacle to sequence-based species identification, and it is manifest that the greatest challenges to biological barcoding will be of taxonomical, rather than technical, nature.

Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data

Summary 1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

The ITS region as a target for characterization of fungal communities using emerging sequencing technologies

The advent of new high-throughput DNA-sequencing technologies promises to redefine the way in which fungi and fungal communities--as well as other groups of organisms--are studied in their natural environment. With read lengths of some few hundred base pairs, massively parallel sequencing (pyrosequencing) stands out among the new technologies as the most apt for large-scale species identification in environmental samples. Although parallel pyrosequencing can generate hundreds of thousands of sequences at an exceptional speed, the limited length of the reads may pose a problem to the species identification process. This study explores whether the discrepancy in read length between parallel pyrosequencing and traditional (Sanger) sequencing will have an impact on the perceived taxonomic affiliation of the underlying species. Based on all 39,200 publicly available fungal environmental DNA sequences representing the nuclear ribosomal internal transcribed spacer (ITS) region, the results show that the two approaches give rise to quite different views of the diversity of the underlying samples. Standardization of which subregion from the ITS region should be sequenced, as well as a recognition that the composition of fungal communities as depicted through different sequencing methods need not be directly comparable, appear crucial to the integration of the new sequencing technologies with current mycological praxis.

ShotgunFunctionalizeR: an R-package for functional comparison of metagenomes

UNLABELLED Microorganisms are ubiquitous in nature and constitute intrinsic parts of almost every ecosystem. A culture-independent and powerful way to study microbial communities is metagenomics. In such studies, functional analysis is performed on fragmented genetic material from multiple species in the community. The recent advances in high-throughput sequencing have greatly increased the amount of data in metagenomic projects. At present, there is an urgent need for efficient statistical tools to analyse these data. We have created ShotgunFunctionalizeR, an R-package for functional comparison of metagenomes. The package contains tools for importing, annotating and visualizing metagenomic data produced by shotgun high-throughput sequencing. ShotgunFunctionalizeR contains several statistical procedures for assessing functional differences between samples, both for individual genes and for entire pathways. In addition to standard and previously published methods, we have developed and implemented a novel approach based on a Poisson model. This procedure is highly flexible and thus applicable to a wide range of different experimental designs. We demonstrate the potential of ShotgunFunctionalizeR by performing a regression analysis on metagenomes sampled at multiple depths in the Pacific Ocean. AVAILABILITY http://shotgun.zool.gu.se

Mining metadata from unidentified ITS sequences in GenBank: A case study in Inocybe (Basidiomycota)

BackgroundThe lack of reference sequences from well-identified mycorrhizal fungi often poses a challenge to the inference of taxonomic affiliation of sequences from environmental samples, and many environmental sequences are thus left unidentified. Such unidentified sequences belonging to the widely distributed ectomycorrhizal fungal genus Inocybe (Basidiomycota) were retrieved from GenBank and divided into species that were identified in a phylogenetic context using a reference dataset from an ongoing study of the genus. The sequence metadata of the unidentified Inocybe sequences stored in GenBank, as well as data from the corresponding original papers, were compiled and used to explore the ecology and distribution of the genus. In addition, the relative occurrence of Inocybe was contrasted to that of other mycorrhizal genera.ResultsMost species of Inocybe were found to have less than 3% intraspecific variability in the ITS2 region of the nuclear ribosomal DNA. This cut-off value was used jointly with phylogenetic analysis to delimit and identify unidentified Inocybe sequences to species level. A total of 177 unidentified Inocybe ITS sequences corresponding to 98 species were recovered, 32% of which were successfully identified to species level in this study. These sequences account for an unexpectedly large proportion of the publicly available unidentified fungal ITS sequences when compared with other mycorrhizal genera. Eight Inocybe species were reported from multiple hosts and some even from hosts forming arbutoid or orchid mycorrhizae. Furthermore, Inocybe sequences have been reported from four continents and in climate zones ranging from cold temperate to equatorial climate. Out of the 19 species found in more than one study, six were found in both Europe and North America and one was found in both Europe and Japan, indicating that at least many north temperate species have a wide distribution.ConclusionAlthough DNA-based species identification and circumscription are associated with practical and conceptual difficulties, they also offer new possibilities and avenues for research. Metadata assembly holds great potential to synthesize valuable information from community studies for use in a species and taxonomy-oriented framework.

An outlook on the fungal internal transcribed spacer sequences in GenBank and the introduction of a web-based tool for the exploration of fungal diversity.

The environmental and distributional data associated with fungal internal transcribed spacer (ITS) sequences in GenBank are investigated and a new web-based tool with which these sequences can be explored is introduced. All fungal ITS sequences in GenBank were classified as either identified to species level or insufficiently identified and compared using BLAST. The results are made available as a biweekly updated web service that can be queried to retrieve all insufficiently identified sequences (IIS) associated with any fungal genus. The most commonly available annotation items in GenBank are isolation source (55%); country of origin (50%); and specific host (38%). The molecular sampling of fungi shows a bias towards North America, Europe, China, and Japan whereas vast geographical areas remain effectively unexplored. Mycorrhizal and parasitic genera are on average associated with more IIS than are saprophytic taxa. Glomus, Alternaria, and Tomentella are the genera represented by the highest number of insufficiently identified ITS sequences in GenBank. The web service presented (http://andromeda.botany.gu.se/emerencia.html#genus_search) offers new means, particularly for mycorrhizal and plant pathogenic fungi, to examine the IIS in GenBank in a taxon-oriented framework and to explore their metadata in an easily accessible and time-efficient manner.

BacMet: antibacterial biocide and metal resistance genes database

Antibiotic resistance has become a major human health concern due to widespread use, misuse and overuse of antibiotics. In addition to antibiotics, antibacterial biocides and metals can contribute to the development and maintenance of antibiotic resistance in bacterial communities through co-selection. Information on metal and biocide resistance genes, including their sequences and molecular functions, is, however, scattered. Here, we introduce BacMet (http://bacmet.biomedicine.gu.se)—a manually curated database of antibacterial biocide- and metal-resistance genes based on an in-depth review of the scientific literature. The BacMet database contains 470 experimentally verified resistance genes. In addition, the database also contains 25 477 potential resistance genes collected from public sequence repositories. All resistance genes in the BacMet database have been organized according to their molecular function and induced resistance phenotype.

A software pipeline for processing and identification of fungal ITS sequences

BackgroundFungi from environmental samples are typically identified to species level through DNA sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region for use in BLAST-based similarity searches in the International Nucleotide Sequence Databases. These searches are time-consuming and regularly require a significant amount of manual intervention and complementary analyses. We here present software – in the form of an identification pipeline for large sets of fungal ITS sequences – developed to automate the BLAST process and several additional analysis steps. The performance of the pipeline was evaluated on a dataset of 350 ITS sequences from fungi growing as epiphytes on building material.ResultsThe pipeline was written in Perl and uses a local installation of NCBI-BLAST for the similarity searches of the query sequences. The variable subregion ITS2 of the ITS region is extracted from the sequences and used for additional searches of higher sensitivity. Multiple alignments of each query sequence and its closest matches are computed, and query sequences sharing at least 50% of their best matches are clustered to facilitate the evaluation of hypothetically conspecific groups. The pipeline proved to speed up the processing, as well as enhance the resolution, of the evaluation dataset considerably, and the fungi were found to belong chiefly to the Ascomycota, with Penicillium and Aspergillus as the two most common genera. The ITS2 was found to indicate a different taxonomic affiliation than did the complete ITS region for 10% of the query sequences, though this figure is likely to vary with the taxonomic scope of the query sequences.ConclusionThe present software readily assigns large sets of fungal query sequences to their respective best matches in the international sequence databases and places them in a larger biological context. The output is highly structured to be easy to process, although it still needs to be inspected and possibly corrected for the impact of the incomplete and sometimes erroneously annotated fungal entries in these databases. The open source pipeline is available for UNIX-type platforms, and updated releases of the target database are made available biweekly. The pipeline is easily modified to operate on other molecular regions and organism groups.