Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation

Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database—currently the most complete database for lichen-forming fungi—can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

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Genome-Directed Isolation of the Key Nitrogen Fixer Leptospirillum ferrodiazotrophum sp. nov. from an Acidophilic Microbial Community

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.6319-6324.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 6319-6324 ◽

Cited By ~ 169

Author(s):

Gene W. Tyson ◽

Ian Lo ◽

Brett J. Baker ◽

Eric E. Allen ◽

Philip Hugenholtz ◽

...

Keyword(s):

Mine Drainage ◽

Sequence Data ◽

Internal Transcribed Spacer Region ◽

Free Living ◽

Maximum Cell Density ◽

Group Iii ◽

A Genome ◽

Low Diversity ◽

Maximum Cell ◽

Uncultured Microorganisms

ABSTRACT Analysis of assembled random shotgun sequence data from a low-diversity, subsurface acid mine drainage (AMD) biofilm revealed a single nif operon. This was found on a genome fragment belonging to a member of Leptospirillum group III, a lineage in the Nitrospirae phylum with no cultivated representatives. Based on the prediction that this organism is solely responsible for nitrogen fixation in the community, we pursued a selective isolation strategy to obtain the organism in pure culture. An AMD biofilm sample naturally abundant in Leptospirillum group III cells was homogenized, filtered, and serially diluted into a nitrogen-free liquid medium. The resulting culture in the terminal dilution grew autotrophically to a maximum cell density of ∼106 cells/ml, oxidizing ferrous iron as the sole energy source. 16S rRNA-internal transcribed spacer region clone library analysis confirmed that the isolate is a member of Leptospirillum group III and that the culture is axenic. We propose the name Leptospirillum ferrodiazotrophum sp. nov. for this iron-oxidizing, free-living diazotroph. This study highlights how environmental sequence data can provide insights for culturing previously uncultured microorganisms.

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Metagenomic data reveal diverse fungal and algal communities associated with the lichen symbiosis

10.1101/2020.03.04.966853 ◽

2020 ◽

Cited By ~ 2

Author(s):

Hayden Smith ◽

Francesco Dal Grande ◽

Lucia Muggia ◽

Rachel Keuler ◽

Pradeep K. Divakar ◽

...

Keyword(s):

Dna Barcode ◽

Metagenomic Data ◽

Future Research ◽

Internal Transcribed Spacer Region ◽

Algal Communities ◽

Lichen Symbiosis ◽

Fungal Partner ◽

Wide Range ◽

Symbiotic Phenotype ◽

Taxonomic Assignments

AbstractLichens have traditionally been considered the symbiotic phenotype from the interactions of a single fungal partner and one or few photosynthetic partners. However, the lichen symbiosis has been shown to be far more complex and may include a wide range of other interacting organisms, including non-photosynthetic bacteria, accessory fungi, and algae. In this study, we analyzed metagenomic shotgun sequences to better characterize lichen mycobiomes. Specifically, we inferred the range of fungi associated within lichen thalli from five groups of lichens – horsehair lichens (mycobiont=Bryoria spp.), shadow lichens (taxa in Physciaceae), rock posies (Rhizoplaca spp.), rock tripes (Umbilicaria spp.), and green rock shields (Xanthoparmelia spp.). Metagenomic reads from the multi-copy nuclear ribosomal internal transcribed spacer region, the standard DNA barcode region for fungi, were extracted, clustered, and used to infer taxonomic assignments. Our data revealed diverse lichen-associated mycobiomes, and closely related mycobionts tended to have more similar mycobiomes. Many of the members of the lichen-associated mycobiomes identified here have not previously been found in association with lichens. We found little evidence supporting the ubiquitous presence of Cystobasidiales yeasts in macrolichens, although reads representing this putative symbiotic partner were found in samples of horsehair lichens, albeit in low abundance. Our study further highlights the ecosystem-like features of lichens, with partners and interactions far from being completely understood. Future research is needed to more fully and accurately characterize lichen mycobiomes and how these fungi interact with the major lichen components – the photo- and mycobionts.

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Fungal-algal interactions in Ramalina menziesii and its associated epiphytic lichen community

The Lichenologist ◽

10.1017/s0024282912000138 ◽

2012 ◽

Vol 44 (4) ◽

pp. 543-560 ◽

Cited By ~ 12

Author(s):

Silke WERTH

Keyword(s):

Dna Sequences ◽

Species Interactions ◽

Phylogenetic Trees ◽

Algal Species ◽

Fungal Species ◽

Complex Nature ◽

Internal Transcribed Spacer Region ◽

Epiphytic Lichen ◽

Lichen Community ◽

Symbiotic Mutualism

AbstractLichens are a fascinating example of a symbiotic mutualism. It is still uncertain which processes guide fungal-photobiont interactions, and whether they are random or of a more complex nature. Here, the fungal-algal interactions in Ramalina menziesii and co-occurring taxa are analyzed by using DNA sequences of the algal Internal Transcribed Spacer region (ITS), to investigate fungal-algal associations in juvenile R. menziesii and allied species. Algal species were identified by a combination of BLAST searches, median-joining network analysis, and Bayesian phylogenetics. Fungal-algal networks were analyzed for nestedness, both at the species and haplotype level (fungal species vs. algal haplotypes), and the networks were inspected for evidence of compartmentalization. Bayesian phylogenetic trees indicated that the widespread green alga Trebouxia decolorans associated with R. menziesii, as well as six other fungal species. Four additional fungal species interacted with four different species of Trebouxia. Only in one out of ten samples were algal haplotypes shared with the nearest neighbours of juvenile R. menziesii. Fungal-algal species interactions were compartmentalized, while at the level of algal haplotypes, nestedness was found. This pattern is similar to the compartmentalization found in other intimately interacting mutualists.

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Field-based species identification in eukaryotes using real-time nanopore sequencing

10.1101/107656 ◽

2017 ◽

Cited By ~ 1

Author(s):

Joe Parker ◽

Andrew J. Helmstetter ◽

Dion Devey ◽

Alexander S.T. Papadopulos

Keyword(s):

Real Time ◽

Species Identification ◽

Dna Sequences ◽

High Throughput Sequencing ◽

De Novo ◽

Reference Database ◽

Nanopore Sequencing ◽

Technological Advances ◽

A Genome ◽

Hybrid Genome

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored1,2. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere3–5. However, the generation of datasets from large, complex genomes has been constrained to laboratories6,7. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.)8 has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification-by-sequencing and de novo phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, “al fresco” laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible. These technological advances are set to revolutionise research in the biological sciences9 and have broad implications for conservation, taxonomy, border agencies and citizen science.

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Kaiju: Fast and sensitive taxonomic classification for metagenomics

10.1101/031229 ◽

2015 ◽

Cited By ~ 7

Author(s):

Peter Menzel ◽

Kim Lee Ng ◽

Anders Krogh

Keyword(s):

Large Scale ◽

Taxonomic Classification ◽

Greedy Heuristic ◽

Reference Database ◽

The Novel ◽

Sequencing Technologies ◽

A Genome ◽

Reference Databases ◽

Genome Exclusion ◽

Higher Sensitivity

The constantly decreasing cost and increasing output of current sequencing technologies enable large scale metagenomic studies of microbial communities from diverse habitats. Therefore, fast and accurate methods for taxonomic classification are needed, which can operate on increasingly larger datasets and reference databases. Recently, several fast metagenomic classifiers have been developed, which are based on comparison of genomic k-mers. However, nucleotide comparison using a fixed k-mer length often lacks the sensitivity to overcome the evolutionary distance between sampled species and genomes in the reference database. Here, we present the novel metagenome classifier Kaiju for fast assignment of reads to taxa. Kaiju finds maximum exact matches on the protein-level using the Borrows-Wheeler transform, and can optionally allow amino acid substitutions in the search using a greedy heuristic. We show in a genome exclusion study that Kaiju can classify more reads with higher sensitivity and similar precision compared to fast k-mer based classifiers, especially in genera that are underrepresented in reference databases. We also demonstrate that Kaiju classifies more than twice as many reads in ten real metagenomes compared to programs based on genomic k-mers. Kaiju can process up to millions of reads per minute, and its memory footprint is below 6 GB of RAM, allowing the analysis on a standard PC. The program is available under the GPL3 license at: http://bioinformatics-centre.github.io/kaiju

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Pythium spp. Associated with Rooibos Seedlings, and Their Pathogenicity Toward Rooibos, Lupin, and Oat

Plant Disease ◽

10.1094/pdis-05-13-0467-re ◽

2014 ◽

Vol 98 (2) ◽

pp. 223-232 ◽

Cited By ~ 9

Author(s):

Amirhossein Bahramisharif ◽

Sandra C. Lamprecht ◽

Christoffel F. J. Spies ◽

Wilhelm J. Botha ◽

Frikkie J. Calitz ◽

...

Keyword(s):

Phytophthora Cinnamomi ◽

Phylogenetic Analyses ◽

Common Species ◽

Limited Information ◽

Internal Transcribed Spacer Region ◽

Pythium Irregulare ◽

Rotation Crops ◽

Native Site ◽

Highly Virulent ◽

Pythium Acanthicum

Rooibos (Aspalathus linearis) is an important indigenous crop in South Africa. Oomycetes are a common problem in rooibos nurseries, causing serious losses, but limited information is available on the species involved. Molecular and morphological analyses of 117 oomycete isolates from 19 rooibos nurseries and 33 isolates from 11 native rooibos sites revealed the presence of several Pythium spp., including Pythium acanthicum, P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, P. cederbergense, and Pythium RB II, and Phytophthora cinnamomi (native site). Most of the species were identified in nurseries and native rooibos, with Pythium irregulare being the most common species occurring in all nurseries and 46% of the native sites. Phylogenetic analyses of the internal transcribed spacer region of the P. irregulare isolates showed that isolates within this species complex fit into three subclades, of which only two have previously been reported. On rooibos, all species except P. acanthicum and the previously characterized P. cederbergense and Pythium RB II were pathogenic and highly virulent. On lupin and oat, rotation crops in nurseries, the three aforementioned species were also nonpathogenic. All the other oomycete species were pathogenic on lupin but less so than on rooibos. On oat, only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic. This is the first report of P. mamillatum, P. pyrilobum, and P. myriotylum as pathogens of lupin, and P. irregulare and P. pyrilobum as pathogens of oat. The three nonpathogenic Pythium spp. were able to significantly reduce disease caused by pathogenic species in the less susceptible lupin and oat but not on rooibos. On lupin, the nonpathogenic species enhanced the virulence of Phytophthora cinnamomi.

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Needlestack: an ultra-sensitive variant caller for multi-sample next generation sequencing data

10.1101/639377 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tiffany M. Delhomme ◽

Patrice H. Avogbe ◽

Aurélie Gabriel ◽

Nicolas Alcala ◽

Noemie Leblay ◽

...

Keyword(s):

Next Generation Sequencing ◽

Error Rate ◽

Somatic Mutations ◽

Next Generation Sequencing Data ◽

Sequencing Error ◽

Next Generation ◽

Sequencing Error Rate ◽

Main Challenge ◽

A Genome ◽

Generation Sequencing

ABSTRACTThe emergence of Next-Generation Sequencing (NGS) has revolutionized the way of reaching a genome sequence, with the promise of potentially providing a comprehensive characterization of DNA variations. Nevertheless, detecting somatic mutations is still a difficult problem, in particular when trying to identify low abundance mutations such as subclonal mutations, tumour-derived alterations in body fluids or somatic mutations from histological normal tissue. The main challenge is to precisely distinguish between sequencing artefacts and true mutations, particularly when the latter are so rare they reach similar abundance levels as artefacts. Here, we present needlestack, a highly sensitive variant caller, which directly learns from the data the level of systematic sequencing errors to accurately call mutations. Needlestack is based on the idea that the sequencing error rate can be dynamically estimated from analyzing multiple samples together. We show that the sequencing error rate varies across alterations, illustrating the need to precisely estimate it. We evaluate the performance of needlestack for various types of variations, and we show that needlestack is robust among positions and outperforms existing state-of-the-art method for low abundance mutations. Needlestack, along with its source code is freely available on the GitHub plateform: https://github.com/IARCbioinfo/needlestack.

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From DNA sequences to operational reference databases: an opinionated approach using R

ARPHA Conference Abstracts ◽

10.3897/aca.4.e64936 ◽

2021 ◽

Vol 4 ◽

Author(s):

François Keck ◽

Florian Altermatt

Keyword(s):

Dna Sequences ◽

Relational Databases ◽

Reference Database ◽

Complex Data ◽

Biological Sequence ◽

R Language ◽

Speed Up ◽

Reference Databases ◽

Taxonomic Groups ◽

User Friendly

Reference databases of sequences that have been taxonomically assigned are a key element for DNA-based identification of organisms. Accurate and complete reference databases are necessary to associate a correct taxonomic name to the sequences obtained in studies using metabarcoding. Today many research projects using DNA metabarcoding include the development of a custom reference database, often derived from large repositories like GenBank. At the same time, many projects are focussing on the development of ready-to-use databases validated by experts and targeting specific markers and taxonomic groups. While mainstream tools such as spreadsheet softwares may be suitable to manage small databases, they quickly become insufficient when the amount of data increases and validation operations become more complex. There is a clear need for providing user‐friendly and powerful tools to manipulate biological sequences and manage reference databases. The R language which is a free software and has already been adopted by many researchers to perform their analyses is highly suitable to develop such tools. In this talk, we will outline the approach we recommend to handle small- to middle-sized reference databases, currently still making the majority of projects. We will advocate that a simple tabular approach where each sequence constitutes an observation may be the most adequate. While such a single table may be less flexible and less optimized than relational databases or more complex data structures, it is easy to maintain and allows the direct use of modern dataframe centric tools. We will specifically present and discuss two R packages that can be used jointly to make reference database development more accessible and more reproducible. First, we will briefly introduce bioseq (Keck 2020) which is dedicated to biological sequence manipulation and analysis. The package implements classes and functions to make analyses of complex datasets including DNA, RNA or protein sequences as simple as possible. The strength of bioseq is to provide standard and more advanced functions to perform low level operations through a simple and consistent programming interface. Then we will present refdb, which has been developed as an environment for semi-automatic and assisted construction of reference databases. The refdb package is a reference database manager offering a set of powerful functions to import, organize, clean, filter, audit and export the data. We will outline how these two packages together can speed up reference database generation and handling, and contribute to standardization and repeatability in metabarcoding studies.

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Incorporating genome-based phylogeny and trait similarity into diversity assessments helps to resolve a global collection of human gut metagenomes

10.1101/2020.07.16.207845 ◽

2020 ◽

Author(s):

Nicholas D. Youngblut ◽

Jacobo de la Cuesta-Zuluaga ◽

Ruth E. Ley

Keyword(s):

Microbial Communities ◽

Large Scale ◽

Alpha Diversity ◽

Reference Database ◽

Rrna Gene ◽

Human Gut ◽

Shotgun Metagenomics ◽

Diversity Measures ◽

Microbiome Diversity ◽

A Genome

AbstractTree-based diversity measures incorporate phylogenetic or phenotypic relatedness into comparisons of microbial communities. This improves the identification of explanatory factors compared to tree-agnostic diversity measures. However, applying tree-based diversity measures to metagenome data is more challenging than for single-locus sequencing (e.g., 16S rRNA gene). The Genome Taxonomy Database (GTDB) provides a genome-based reference database that can be used for species-level metagenome profiling, and a multi-locus phylogeny of all genomes that can be employed for diversity calculations. Moreover, traits can be inferred from the genomic content of each representative, allowing for trait-based diversity measures. Still, it is unclear how metagenome-based assessments of microbiome diversity benefit from incorporating phylogeny or phenotype into measures of diversity. We assessed this by measuring phylogeny-based, trait-based, and tree-agnostic diversity measures from a large, global collection of human gut metagenomes composed of 33 studies and 3348 samples. We found phylogeny- and trait-based alpha diversity to better differentiate samples by westernization, age, and gender. PCoA ordinations of phylogeny- or trait-based weighted UniFrac explained more variance than tree-agnostic measures, which was largely a result of these measures emphasizing inter-phylum differences between Bacteroidaceae (Bacteroidota) and Enterobacteriaceae (Proteobacteria) versus just differences within Bacteroidaceae (Bacteroidota). The disease state of samples was better explained by tree-based weighted UniFrac, especially the presence of Shiga toxin-producing E. coli (STEC) and hypertension. Our findings show that metagenome diversity estimation benefits from incorporating a genome-derived phylogeny or traits.ImportanceEstimations of microbiome diversity are fundamental to understanding spatiotemporal changes of microbial communities and identifying which factors mediate such changes. Tree-based measures of diversity are widespread for amplicon-based microbiome studies due to their utility relative to tree-agnostic measures; however, tree-based measures are seldomly applied to shotgun metagenomics data. We evaluated the utility of phylogeny-, trait-, and tree-agnostic diversity measures on a large scale human gut metagenome dataset to help guide researchers with the complex task of evaluating microbiome diversity via metagenomics.

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Epiphytic lichens of tree-line forests in the Central-Eastern Italian Alps and their importance for conservation

The Lichenologist ◽

10.1017/s0024282906006220 ◽

2006 ◽

Vol 38 (4) ◽

pp. 373-382 ◽

Cited By ~ 9

Author(s):

Juri NASCIMBENE ◽

Stefano MARTELLOS ◽

Pier Luigi NIMIS

Keyword(s):

Species Richness ◽

Rare Species ◽

Common Species ◽

Tree Line ◽

Italian Alps ◽

Epiphytic Lichen ◽

Number Of Species ◽

Subalpine Belt ◽

Large Trees ◽

Central Eastern

135 infrageneric taxa of epiphytic lichen were found in 20 stands of tree-line forests of the Central-Eastern Italian Alps. Three forest types were considered: (1) late successional stands with several large trees, (2) pioneer stands on abandoned pastures without large trees, and (3) open and grazed stands. They were compared on the basis of four main criteria: (1) species richness, (2) number of rare species, (3) number of species that are exclusive to the subalpine belt in Italy, and (4) number of species that are exclusive to the Alps in Italy. Species richness is higher in the late successional stands, which also host a higher share of rare and exclusive species. The total number of rare species per site is correlated with the total number of species, as well as with the number of common species, and with the total number of macrolichens. Rare macrolichens are correlated with common macrolichens. Two main groups of target species with decreasing conservation priority are identified. Letharia vulpina is suggested as signal species for sites worthy of conservation. The guild of macrolichens may be used as an indicator of both species richness and of the occurrence of rare species.

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