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 High-throughput amplicon sequencing-based analysis of active fungal communities inhabiting grapevine after hot-water treatments reveals unexpectedly high fungal diversity

2018 ◽  
Vol 36 ◽  
pp. 26-38 ◽  
Author(s):  
Ales Eichmeier ◽  
Jakub Pečenka ◽  
Eliska Peňázová ◽  
Miroslav Baránek ◽  
Santiago Català-García ◽  
...  
Keyword(s):  
High Throughput ◽  
Fungal Diversity ◽  
Amplicon Sequencing ◽  
Hot Water ◽  
Fungal Communities ◽  
Water Treatments

scholarly journals Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Jaime Aguayo ◽  
Céline Fourrier-Jeandel ◽  
Claude Husson ◽  
Renaud Ioos
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Linear Models ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Correct Identification ◽  
Spore Trap

ABSTRACTTechniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCEThe advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

scholarly journals Metatranscriptomics as a tool to identify fungal species and subspecies in mixed communities

2019 ◽  
Author(s):  
Vanesa R. Marcelino ◽  
Laszlo Irinyi ◽  
John-Sebastian Eden ◽  
Wieland Meyer ◽  
Edward C. Holmes ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Dna Barcode ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Strain Level ◽  
Base Line ◽  
Mock Community ◽  
Culture Independent ◽  
Mixed Communities

AbstractHigh-throughput sequencing (HTS) enables the generation of large amounts of genome sequence data at a reasonable cost. Organisms in mixed microbial communities can now be sequenced and identified in a culture-independent way, usually using amplicon sequencing of a DNA barcode. Bulk RNA-seq (metatranscriptomics) has several advantages over DNA-based amplicon sequencing: it is less susceptible to amplification biases, it captures only living organisms, and it enables a larger set of genes to be used for taxonomic identification. Using a defined mock community comprised of 17 fungal isolates, we evaluated whether metatranscriptomics can accurately identify fungal species and subspecies in mixed communities. Overall, 72.9% of the RNA transcripts were classified, from which the vast majority (99.5%) were correctly identified at the species-level. Of the 15 species sequenced, 13 were retrieved and identified correctly. We also detected strain-level variation within theCryptococcusspecies complexes: 99.3% of transcripts assigned toCryptococcuswere classified as one of the four strains used in the mock community. Laboratory contaminants and/or misclassifications were diverse but represented only 0.44% of the transcripts. Hence, these results show that it is possible to obtain accurate species- and strain-level fungal identification from metatranscriptome data as long as taxa identified at low abundance are discarded to avoid false-positives derived from contamination or misclassifications. This study therefore establishes a base-line for the application of metatranscriptomics in clinical mycology and ecological studies.

Preliminary analysis of amplicon high-throughput sequencing as a method for the assessment of fungal diversity in discolored wood

Holzforschung ◽  
2017 ◽  
Vol 71 (10) ◽  
pp. 793-800 ◽  
Author(s):  
Xingxia Ma ◽  
Mingliang Jiang ◽  
Junliang Liu ◽  
Hao Deng ◽  
Shuangyong Wang
Keyword(s):  
High Throughput ◽  
Internal Transcribed Spacer ◽  
Fungal Diversity ◽  
Preliminary Analysis ◽  
High Throughput Sequencing ◽  
Fungal Species ◽  
Quantitative Relation ◽  
Core Microbiome ◽  
Microbiome Analysis ◽  
First Time

AbstractThe diversity of stain fungi is important if wood is inhabited with various fungi, and the discoloration mechanism will be better understood. MiSeq amplicon high-throughput sequencing (Illumina®) is able to detect species richness (the number of species within a community) and species evenness (the sizes of species populations within a community). This study detected fungal diversity in discolored Mongolian pine for the first time by the MiSeq approach, focusing on the nuclear ribosomal internal transcribed spacer-1 (ITS1). The results show that the discolored wood was inhabited by a combination of microorganisms, more than 90% of which belong toAscomycotafungi at the phylum level. The MiSeq method revealed not only all the inhabited fungal species but also their quantitative relation to each other. The dominant fungal species in sample A areHelotiales(34.1%) andHypocreales(20.7%). The dominant fungal species in sample B isNectriaceae(67.9%), whileHypocrea(34.7%) andSporothrix(27.6%) are the dominant fungal species in sample C. It was confirmed via core microbiome analysis that the following fungi were common stain fungi in the three discolored samples:Fusariumspp.,Aspergillusspp.,Sporothrixspp.,Penicilliumspp.,Trichodermaspp.,Alternariaspp. andCladophialophoraspp.

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.

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing

2020 ◽  
Vol 319 ◽  
pp. 108496 ◽  
Author(s):  
Mengyue Guo ◽  
Wenjun Jiang ◽  
Meihua Yang ◽  
Xiaowen Dou ◽  
Xiaohui Pang
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Fungal Communities

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.

High-throughput sequencing analysis of endophytic fungal diversity in cynanchum sp.

2020 ◽  
Vol 134 ◽  
pp. 349-358
Author(s):  
W.-H. Chen ◽  
S.-J. Wu ◽  
X.-L. Sun ◽  
K.-M. Feng ◽  
K. Rahman ◽  
...  
Keyword(s):  
High Throughput ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Sequencing Analysis

scholarly journals Fungal Community Analysis and Biodeterioration of Waterlogged Wooden Lacquerware from the Nanhai No. 1 Shipwreck

2020 ◽  
Vol 10 (11) ◽  
pp. 3797
Author(s):  
Yin Jia ◽  
Liuyu Yin ◽  
Fengyu Zhang ◽  
Mei Wang ◽  
Mingliang Sun ◽  
...  
Keyword(s):  
Fusarium Solani ◽  
High Throughput Sequencing ◽  
Environmental Parameters ◽  
Community Analysis ◽  
Fungal Species ◽  
Potato Dextrose Agar ◽  
Fungal Communities ◽  
Cellulolytic Enzymes ◽  
Biocidal Products

To avoid the lacquerware of the Nanhai No. 1 shipwreck from being corroded by microorganisms and to improve the knowledge on microbial ecology of the wood lacquers, we conducted a series of tests on the two water samples storing the lacquerware and colonies on the surface of the lacquerware. The high-throughput sequencing detected dominant fungal communities. After that, the fungal strains were isolated and then identified by amplification of ITS- 18S rRNA. Then the activity of ligninolytic and cellulolytic enzymes was detected on potato dextrose agar (PDA) plates with 0.04% (v/v) guaiacol and carboxymethyl cellulose (CMC) agar plates. Finally, we tested the biocide susceptibility of these fungi. Penicillium chrysogenum (NK-NH3) and Fusarium solani (NK- NH1) were the dominant fungi in the sample collected in April 2016 and June 2017. What is more, both showed activity of ligninolytic and cellulolytic enzymes. Four biocidal products (Preventol® D7, P91, BIT 20N, and Euxyl® K100) inhibited the growth of the fungal species in vitro effectively. In further research, the microbial community and environmental parameters in the museum should be monitored to assess the changes in the community and to detect potential microbial outbreaks.

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