scholarly journals Culture-Dependent and Amplicon Sequencing Approaches Reveal Diversity and Distribution of Black Fungi in Antarctic Cryptoendolithic Communities

2021 ◽  
Vol 7 (3) ◽  
pp. 213
Author(s):  
Laura Selbmann ◽  
Gerardo A. Stoppiello ◽  
Silvano Onofri ◽  
Jason E. Stajich ◽  
Claudia Coleine
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Physical Stability ◽  
Environmental Parameters ◽  
Sun Exposure ◽  
Amplicon Sequencing ◽  
Life Forms ◽  
Black Fungi ◽  
The Antarctic ◽  
Culture Dependent

In the harshest environmental conditions of the Antarctic desert, normally incompatible with active life, microbes are adapted to exploit the cryptoendolithic habitat (i.e., pore spaces of rocks) and represent the predominant life-forms. In the rocky niche, microbes take advantage of the thermal buffering, physical stability, protection against UV radiation, excessive solar radiation, and water retention—of paramount importance in one of the driest environments on Earth. In this work, high-throughput sequencing and culture-dependent approaches have been combined, for the first time, to untangle the diversity and distribution of black fungi in the Antarctic cryptoendolithic microbial communities, hosting some of the most extreme-tolerant microorganisms. Rock samples were collected in a vast area, along an altitudinal gradient and opposite sun exposure—known to influence microbial diversity—with the aim to compare and integrate results gained with the two approaches. Among black fungi, Friedmanniomyces endolithicus was confirmed as the most abundant taxon. Despite the much stronger power of the high-throughput sequencing, several species were not retrieved with DNA sequencing and were detectable by cultivation only. We conclude that both culture-dependent and -independent analyses are needed for a complete overview of black fungi diversity. The reason why some species remain undetectable with molecular methods are speculated upon. The effect of environmental parameters such as sun exposure on relative abundance was clearer if based on the wider biodiversity detected with the molecular approach.

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Diversity of dinoflagellate assemblages in coastal temperate and offshore tropical waters of Australia

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tahnee Manning ◽  
Arjun Venkatesh Thilagaraj ◽  
Dmitri Mouradov ◽  
Richard Piola ◽  
Clare Grandison ◽  
...  
Keyword(s):  
Dissolved Oxygen ◽  
High Throughput ◽  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
High Throughput Sequencing ◽  
Environmental Parameters ◽  
Marine Phytoplankton ◽  
Tropical Waters ◽  
Total Sequence ◽  
Phytoplankton Communities

Abstract Background Dinoflagellates are a ubiquitous and ecologically important component of marine phytoplankton communities, with particularly notable species including those associated with harmful algal blooms (HABs) and those that bioluminesce. High-throughput sequencing offers a novel approach compared to traditional microscopy for determining species assemblages and distributions of dinoflagellates, which are poorly known especially in Australian waters. Results We assessed the composition of dinoflagellate assemblages in two Australian locations: coastal temperate Port Phillip Bay and offshore tropical waters of Davies Reef (Great Barrier Reef). These locations differ in certain environmental parameters reflecting latitude as well as possible anthropogenic influences. Molecular taxonomic assessment revealed more species than traditional microscopy, and it showed statistically significant differences in dinoflagellate assemblages between locations. Bioluminescent species and known associates of HABs were present at both sites. Dinoflagellates in both areas were mainly represented by the order Gymnodiniales (66%—82% of total sequence reads). In the warm waters of Davies Reef, Gymnodiniales were equally represented by the two superclades, Gymnodiniales sensu stricto (33%) and Gyrodinium (34%). In contrast, in cooler waters of Port Phillip Bay, Gymnodiniales was mainly represented by Gyrodinium (82%). In both locations, bioluminescent dinoflagellates represented up to 0.24% of the total sequence reads, with Protoperidinium the most abundant genus. HAB-related species, mainly represented by Gyrodinium, were more abundant in Port Phillip Bay (up to 47%) than at Davies Reef (28%), potentially reflecting anthropogenic influence from highly populated and industrial areas surrounding the bay. The entire assemblage of dinoflagellates, as well as the subsets of HAB and bioluminescent species, were strongly correlated with water quality parameters (R2 = 0.56–0.92). Significant predictors differed between the subsets: HAB assemblages were explained by salinity, temperature, dissolved oxygen, and total dissolved solids; whereas, bioluminescent assemblages were explained only by salinity and dissolved oxygen, and had greater variability. Conclusion High-throughput sequencing and genotyping revealed greater diversity of dinoflagellate assemblages than previously known in both subtropical and temperate Australian waters. Significant correlations of assemblage structure with environmental variables suggest the potential for explaining the distribution and composition of both HAB species and bioluminescent species.

scholarly journals Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

2020 ◽  
Author(s):  
Chuanbo Zhang ◽  
Chao-Hui Ren ◽  
Yan-Li Wang ◽  
Qi-Qi Wang ◽  
Yun-Sheng Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
Fungal Community ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
Bioactive Metabolites ◽  
Fungal Community Structure ◽  
Ophiocordyceps Sinensis ◽  
Culturable Fungi ◽  
Culture Dependent

Abstract Background The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approach. Results A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices (EMC), Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. Total 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, Unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, Unclassified Ascomycota, Unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices (EMC) were significantly distinct from the soil microhabitat (Soil). Meanwhile, seven culture media that benefit for the growth of O. sinensis were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains isolated for rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora, Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera of total isolates. Conclusions The significantly distinction and overlap in fungal community structure between two approaches highlight that integration of approaches would generate more information than either of them. Our finding is the first investigation of fungal community structure of natural O. sinensis by two approachs, provide new insight into O. sinensis associated fungi, and support that microbiota of O. sinensis is an untapped source for novel bioactive metabolites discovery.

scholarly journals Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

2020 ◽  
Author(s):  
Chuanbo Zhang ◽  
Chao-Hui Ren ◽  
Yan-Li Wang ◽  
Qi-Qi Wang ◽  
Yun-Sheng Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
Fungal Community ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
Comprehensive Understanding ◽  
Fungal Community Structure ◽  
Ophiocordyceps Sinensis ◽  
Culturable Fungi ◽  
Culture Dependent

Abstract Background: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. Results: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices, Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota, unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from the soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16°C, resulted in 77 fungal strains isolated by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. Conclusions: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

scholarly journals Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

2020 ◽  
Author(s):  
Chuanbo Zhang ◽  
Chao-Hui Ren ◽  
Yan-Li Wang ◽  
Qi-Qi Wang ◽  
Yun-Sheng Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
Fungal Community ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
Comprehensive Understanding ◽  
Fungal Community Structure ◽  
Ophiocordyceps Sinensis ◽  
Culturable Fungi ◽  
Culture Dependent

Abstract Background: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. Results: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices (EMC), Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat (Soil), Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota, unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from the soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16°C, resulted in 77 fungal strains isolated by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. Conclusions: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

Shift in the cow milk microbiota during alpine pasture as analyzed by culture dependent and high-throughput sequencing techniques

2020 ◽  
Vol 91 ◽  
pp. 103504 ◽  
Author(s):  
Ilaria Carafa ◽  
Irma Castro Navarro ◽  
Giovanni Bittante ◽  
Franco Tagliapietra ◽  
Luigi Gallo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cow Milk ◽  
Alpine Pasture ◽  
Culture Dependent

scholarly journals Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasemin Guenay-Greunke ◽  
David A. Bohan ◽  
Michael Traugott ◽  
Corinna Wallinger
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Sequencing Depth ◽  
Sequencing Error ◽  
Sequencing Data ◽  
Large Sample ◽  
Sequencing Errors ◽  
Plant Feeding

AbstractHigh-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

scholarly journals Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

2018 ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

AbstractAmplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.ImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

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