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 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 The Fungal Communities and Flavor Profiles in Different Types of High-Temperature Daqu as Revealed by High-Throughput Sequencing and Electronic Senses

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenchao Cai ◽  
Yu’ang Xue ◽  
Yurong Wang ◽  
Wenping Wang ◽  
Na Shu ◽  
...  
Keyword(s):  
High Temperature ◽  
Nitrogen Oxides ◽  
Organic Compounds ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Electronic Tongue ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Significant Difference ◽  
Distinct Character

Polymicrobial co-fermentation is among the distinct character of high-temperature Daqu. However, fungal communities in the three types of high-temperature Daqu, namely, white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, are yet to be characterized. In this study, the fungal diversity, taste, and aroma profiles in the three types of high-temperature Daqu were investigated by Illumina MiSeq high-throughput sequencing, electronic tongue, and electronic nose, respectively. Ascomycota and Basidiomycota were detected as the absolute dominant fungal phylum in all types of high-temperature Daqu samples, whereas Thermomyces, Thermoascus, Aspergillus, Rasamsonia, Byssochlamys, and Trichomonascus were identified as the dominant fungal genera. The fungal communities of the three types of high-temperature Daqu differed significantly (p < 0.05), and Thermomyces, Thermoascus, and Monascus could serve as the biomarkers in white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, respectively. The three types of high-temperature Daqu had an extremely significant difference (p < 0.01) in flavor: white high-temperature Daqu was characterized by sourness, bitterness, astringency, richness, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds; black high-temperature Daqu was characterized by aftertaste-A, aftertaste-B, methane-aliph, hydrogen, and aromatic compounds; and yellow high-temperature Daqu was characterized by saltiness, umami, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds. The fungal communities in the three types of high-temperature Daqu were significantly correlated with taste but not with aroma, and the aroma of high-temperature Daqu was mainly influenced by the dominant fungal genera including Trichomonascus, Aspergillus, Thermoascus, and Thermomyces. The result of the present study enriched and refined our knowledge of high-temperature Daqu, which had positive implications for the development of traditional brewing technique.

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

Analysis of the effects of nanosilver on bacterial community in the intestinal fluid of silkworms using high-throughput sequencing

2019 ◽  
Vol 110 (3) ◽  
pp. 309-320
Author(s):  
Chen Lin ◽  
Zhou Wei ◽  
Zhou Yi ◽  
Tan Tingting ◽  
Du Huamao ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Intestinal Bacteria ◽  
Illumina Miseq ◽  
The Body ◽  
Dependent Manner ◽  
Negative Effects ◽  
Silkworm Larvae ◽  
Intestinal Bacterial Community

AbstractNanosilver is an environment-friendly, harmless alternative of traditional disinfectants which can be potentially applied in the sericulture industry. However, the effects of nanosilver on the intestinal bacterial community of the silkworms (Bombyx mori L.) are unclear. In this study, Illumina MiSeq high-throughput sequencing technology was used to assess the intestinal bacterial community in both male and female silkworms while treated with different concentrations of nanosilver. We found that nanosilver significantly influenced the composition of silkworm intestinal bacterial community on the different taxonomic levels. Most conspicuously, the abundance of Firmicutes was increased by the treatment of 20 mg L−1 nanosilver but decreased by that of 100 mg L−1 nanosilver at the phylum level. The same trend was observed in Bacilli at the class level and in Enterococcus at the genus level. In some extreme cases, application of nanosilver eliminated the bacterium, e.g., Brevibacillus, but increased the population of several other bacteria in the host intestine, such as Blautia, Terrisporobacter, Faecalibacterium, and some bacteria could only be found in nanosilver treatment groups, e.g., Dialister. In addition, although nanosilver generally showed negative effects on the cocooning rate in a dose-dependent manner, we found that 20 mg L−1 nanosilver treatment significantly increased the body weight of silkworms and did not show negative effects on the survival rate. These results indicated that the intestinal bacteria community of silkworm larvae was significantly changed after nanosilver treatment which might consequently influence host growth and development.

scholarly journals DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

2015 ◽  
Vol 105 (6) ◽  
pp. 717-727 ◽  
Author(s):  
G.-J. Brandon-Mong ◽  
H.-M. Gan ◽  
K.-W. Sing ◽  
P.-S. Lee ◽  
P.-E. Lim ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Ease Of Use ◽  
Malaise Trap ◽  
Detection Rates ◽  
Operational Taxonomic Units ◽  
Arthropod Species ◽  
Primer Sets ◽  
Arthropod Biodiversity

AbstractMetabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

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