Influence of DNA extraction and PCR amplification on studies of soil fungal communities based on amplicon sequencing

2011 ◽  
Vol 57 (12) ◽  
pp. 1062-1066 ◽  
Author(s):  
Lihui Xu ◽  
Sabine Ravnskov ◽  
John Larsen ◽  
Mogens Nicolaisen
Keyword(s):  
Species Richness ◽  
Microbial Communities ◽  
Dna Extraction ◽  
454 Pyrosequencing ◽  
Pcr Amplification ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Fungal Communities ◽  
Agricultural Field ◽  
Operational Taxonomic Units

Most studies involving next-generation amplicon sequencing of microbial communities from environmental studies lack replicates. DNA extraction and PCR effects on the variation of read abundances of operational taxonomic units generated from deep amplicon 454 pyrosequencing was investigated using soil samples from an agricultural field with diseased pea. One sample was extracted four times, and one of these samples was PCR amplified four times to obtain eight replicates in total. Results showed that species richness was consistent among replicates. Variation among dominant taxa was low across replicates, whereas rare operational taxonomic units showed higher variation among replicates. The results indicate that pooling of several extractions and PCR amplicons will decrease variation among samples.

Analysis of the structure of bacterial and fungal communities in disease suppressive and disease conducive tobacco-planting soils in China

Soil Research ◽  
2020 ◽  
Vol 58 (1) ◽  
pp. 35
Author(s):  
Lin Gao ◽  
Rui Wang ◽  
Jiaming Gao ◽  
Fangming Li ◽  
Guanghua Huang ◽  
...  
Keyword(s):  
Microbial Communities ◽  
High Throughput Sequencing ◽  
Soil Samples ◽  
Fungal Communities ◽  
Sequencing Technology ◽  
Operational Taxonomic Units ◽  
Tobacco Cultivation ◽  
Bacteria And Fungi ◽  
Disease Suppressive Soil ◽  
Conducive Soil

To clarify the differences between microbial communities resident in disease suppressive soil (DSS) and disease conducive soil (DCS) in tobacco cultivation, representative soil samples were collected from tobacco plantations in Shengjiaba, China, and the structure and diversity of the resident bacterial and fungal communities were analysed using high-throughput sequencing technology. Our results showed a greater number of operational taxonomic units associated with bacteria and fungi in DSS than in DCS. At the phylum level, abundances of Chloroflexi, Saccharibacteria, Firmicutes, and Planctomycetes in DSS were lower than in DCS, but abundance of Gemmatimonadetes was significantly higher. Abundances of Zygomycota and Chytridiomycota were higher in DSS than DCS, but abundance of Rozellomycota was significantly lower. At the genus level, abundances of 18 bacterial and nine fungal genera varied significantly between DSS and DCS. Relative abundances of Acidothermus, Microbacterium, Curtobacterium, and Colletotrichum were higher in DCS than DSS. The Shannon and Chao1 indices of DSS microbial communities were higher than those of DCS communities. High microbial diversity reduces the incidence of soil-borne diseases in tobacco plantations and promotes the formation of DSSs.

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 The response of soil and phyllosphere microbial communities to repeated application of the fungicide iprodione: accelerated biodegradation or toxicity?

2020 ◽  
Vol 96 (6) ◽  
Author(s):  
A Katsoula ◽  
S Vasileiadis ◽  
M Sapountzi ◽  
Dimitrios G Karpouzas
Keyword(s):  
Microbial Communities ◽  
Agricultural Production ◽  
Soil Microorganisms ◽  
Amplicon Sequencing ◽  
Fungal Communities ◽  
Similar Response ◽  
Repeated Application ◽  
Plant Leaves ◽  
Soil System ◽  
Plant Soil

ABSTRACT Pesticides interact with microorganisms in various ways with the outcome being negative or positive for the soil microbiota. Pesticides' effects on soil microorganisms have been studied extensively in soil but not in other pesticides-exposed microbial habitats like the phyllosphere. We tested the hypothesis that soil and phyllosphere support distinct microbial communities, but exhibit a similar response (accelerated biodegradation or toxicity) to repeated exposure to the fungicide iprodione. Pepper plants received four repeated foliage or soil applications of iprodione, which accelerated its degradation in soil (DT50_1st = 1.23 and DT50_4th = 0.48 days) and on plant leaves (DT50_1st > 365 and DT50_4th = 5.95 days). The composition of the epiphytic and soil bacterial and fungal communities, determined by amplicon sequencing, was significantly altered by iprodione. The archaeal epiphytic and soil communities responded differently; the former showed no response to iprodione. Three iprodione-degrading Paenarthrobacter strains were isolated from soil and phyllosphere. They hydrolyzed iprodione to 3,5-dichloraniline via the formation of 3,5-dichlorophenyl-carboxiamide and 3,5-dichlorophenylurea-acetate, a pathway shared by other soil-derived arthrobacters implying a phylogenetic specialization in iprodione biotransformation. Our results suggest that iprodione-repeated application could affect soil and epiphytic microbial communities with implications for the homeostasis of the plant–soil system and agricultural production.

scholarly journals Inter-domain microbial diversity within the coral holobiontSiderastrea sidereafrom two depth habitats

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4323 ◽  
Author(s):  
Guido Bonthond ◽  
Daniel G. Merselis ◽  
Katherine E. Dougan ◽  
Trevor Graff ◽  
William Todd ◽  
...  
Keyword(s):  
Florida Keys ◽  
Amplicon Sequencing ◽  
Fungal Communities ◽  
Physiological Adaptation ◽  
Operational Taxonomic Units ◽  
Clade B ◽  
Holistic Understanding ◽  
Clade C ◽  
Occurrence Patterns ◽  
High Degree

Corals host diverse microbial communities that are involved in acclimatization, pathogen defense, and nutrient cycling. Surveys of coral-associated microbes have been particularly directed towardSymbiodiniumand bacteria. However, a holistic understanding of the total microbiome has been hindered by a lack of analyses bridging taxonomically disparate groups. Using high-throughput amplicon sequencing, we simultaneously characterized theSymbiodinium, bacterial, and fungal communities associated with the Caribbean coralSiderastrea sidereacollected from two depths (17 and 27 m) on Conch reef in the Florida Keys.S. sidereahosted an exceptionally diverseSymbiodiniumcommunity, structured differently between sampled depth habitats. While dominated at 27 m by aSymbiodiniumbelonging to clade C, at 17 mS. sidereaprimarily hosted a mixture of clade B types. Most fungal operational taxonomic units were distantly related to available reference sequences, indicating the presence of a high degree of fungal novelty within theS. sidereaholobiont and a lack of knowledge on the diversity of fungi on coral reefs. Network analysis showed that co-occurrence patterns in theS. sidereaholobiont were prevalent among bacteria, however, also detected between fungi and bacteria. Overall, our data show a drastic shift in the associatedSymbiodiniumcommunity between depths on Conch Reef, which might indicate that alteration in this community is an important mechanism facilitating local physiological adaptation of theS. sidereaholobiont. In contrast, bacterial and fungal communities were not structured differently between depth habitats.

scholarly journals Rbec: a tool for analysis of amplicon sequencing data from synthetic microbial communities

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Pengfan Zhang ◽  
Stjin Spaepen ◽  
Yang Bai ◽  
Stephane Hacquard ◽  
Ruben Garrido-Oter
Keyword(s):  
Microbial Communities ◽  
Amplicon Sequencing ◽  
Fungal Communities ◽  
Polymorphic Variation ◽  
Sequencing Data ◽  
Sequencing Errors ◽  
Extensive Evaluation ◽  
Culture Independent ◽  
Reference Sequences ◽  
Synthetic Microbial Communities

AbstractSynthetic microbial communities (SynComs) constitute an emerging and powerful tool in biological, biomedical, and biotechnological research. Despite recent advances in algorithms for the analysis of culture-independent amplicon sequencing data from microbial communities, there is a lack of tools specifically designed for analyzing SynCom data, where reference sequences for each strain are available. Here we present Rbec, a tool designed for the analysis of SynCom data that accurately corrects PCR and sequencing errors in amplicon sequences and identifies intra-strain polymorphic variation. Extensive evaluation using mock bacterial and fungal communities show that our tool outperforms current methods for samples of varying complexity, diversity, and sequencing depth. Furthermore, Rbec also allows accurate detection of contaminants in SynCom experiments.

scholarly journals Assessing Microbial Communities Related to Mercury Transformations in Contaminated Streambank Soils

2021 ◽  
Vol 232 (1) ◽  
Author(s):  
Yazeed Abdelmageed ◽  
Carrie Miller ◽  
Carrie Sanders ◽  
Timothy Egbo ◽  
Alexander Johs ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Contaminated Soils ◽  
Microbial Community Composition ◽  
Inorganic Mercury ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Rrna Gene ◽  
Enrichment Cultures ◽  
Relative Abundances

AbstractIn nature, the bioaccumulative potent neurotoxin methylmercury (MeHg) is produced from inorganic mercury (Hg) predominantly by anaerobic microorganisms. Hg-contaminated soils are a potential source of MeHg due to microbial activity. We examine streambank soils collected from the contaminated East Fork Poplar Creek (EFPC) in Tennessee, USA, where seasonal variations in MeHg levels have been observed throughout the year, suggesting active microbial Hg methylation. In this study, we characterized the microbial community in contaminated bank soil samples collected from two locations over a period of one year and compared the results to soil samples from an uncontaminated reference site with similar geochemistry (n = 12). Microbial community composition and diversity were assessed by 16S rRNA gene amplicon sequencing. Furthermore, to isolate potential methylators from soils, enrichment cultures were prepared using selective media. A set of three clade-specific primers targeting the gene hgcA were used to detect Hg methylators among the δ-Proteobacteria in EFPC bank soils across all seasons. Two families among the δ-Proteobacteria that have been previously associated with Hg methylation, Geobacteraceae and Syntrophobacteraceae, were found to be predominant with relative abundances of 0.13% and 4.0%, respectively. However, in soil enrichment cultures, Firmicutes were predominant among families associated with Hg methylation. Specifically, Clostridiaceae and Peptococcaceae and their genera Clostridium and Desulfosporosinus were among the ten most abundant genera with relative abundances of 2.6% and 1.7%, respectively. These results offer insights into the role of microbial communities on Hg transformation processes in contaminated bank soils in EFPC. Identifying the biogeochemical drivers of MeHg production is critical for future remediation efforts.

scholarly journals Reference-based error correction of amplicon sequencing data from synthetic communities

2021 ◽  
Author(s):  
Pengfan Zhang ◽  
Stjin Spaepen ◽  
Yang Bai ◽  
Stephane Hacquard ◽  
Ruben Garrido-Oter
Keyword(s):  
Microbial Communities ◽  
Amplicon Sequencing ◽  
R Package ◽  
Fungal Communities ◽  
Polymorphic Variation ◽  
Sequencing Data ◽  
Extensive Evaluation ◽  
Culture Independent ◽  
Reference Sequences ◽  
Synthetic Microbial Communities

AbstractMotivationSynthetic microbial communities (SynComs) constitute an emergent and powerful tool in biological, biomedical, and biotechnological research. Despite recent advances in algorithms for analysis of culture-independent amplicon sequencing data from microbial communities, there is a lack of tools specifically designed for analysing SynCom data, where reference sequences for each strain are available.ResultsHere we present Rbec, a tool designed for analysing SynCom data that outperforms current methods by accurately correcting errors in amplicon sequences and identifying intra-strain polymorphic variation. Extensive evaluation using mock bacterial and fungal communities show that our tool performs robustly for samples of varying complexity, diversity, and sequencing depth. Further, Rbec also allows accurate detection of contaminations in SynCom experiments.AvailabilityRbec is freely available as an open-source R package and can be downloaded at: https://github.com/PengfanZhang/Microbiome.

scholarly journals Microbial communities of poultry house dust, excreta and litter are partially representative of microbiota of chicken caecum and ileum

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255633
Author(s):  
Yugal R. Bindari ◽  
Robert J. Moore ◽  
Thi Thu Hao Van ◽  
Matthew Hilliar ◽  
Shu-Biao Wu ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Ribosomal Rna ◽  
Sampling Methods ◽  
Amplicon Sequencing ◽  
Microbial Composition ◽  
Poultry House ◽  
Operational Taxonomic Units ◽  
Non Invasive ◽  
16S Ribosomal Rna Gene ◽  
Poultry Dust

Traditional sampling methods for the study of poultry gut microbiota preclude longitudinal studies as they require euthanasia of birds for the collection of caecal and ileal contents. Some recent research has investigated alternative sampling methods to overcome this issue. The main goal of this study was to assess to what extent the microbial composition of non-invasive samples (excreta, litter and poultry dust) are representative of invasive samples (caecal and ileal contents). The microbiota of excreta, dust, litter, caecal and ileal contents (n = 110) was assessed using 16S ribosomal RNA gene amplicon sequencing. Of the operational taxonomic units (OTUs) detected in caecal contents, 99.7% were also detected in dust, 98.6% in litter and 100% in excreta. Of the OTUs detected in ileal contents, 99.8% were detected in dust, 99.3% in litter and 95.3% in excreta. Although the majority of the OTUs found in invasive samples were detected in non-invasive samples, the relative abundance of members of the microbial communities of these groups were different, as shown by beta diversity measures. Under the conditions of this study, correlation analysis showed that dust could be used as a proxy for ileal and caecal contents to detect the abundance of the phylum Firmicutes, and excreta as a proxy of caecal contents for the detection of Tenericutes. Similarly, litter could be used as a proxy for caecal contents to detect the abundance of Firmicutes and Tenericutes. However, none of the non-invasive samples could be used to infer the overall abundance of OTUs observed in invasive samples. In conclusion, non-invasive samples could be used to detect the presence and absence of the majority of the OTUs found in invasive samples, but could not accurately reflect the microbial community structure of invasive samples.

scholarly journals Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities

2020 ◽  
Author(s):  
Cecelia Giangacomo ◽  
Mohsen Mohseni ◽  
Lynsey Kovar ◽  
Jason G. Wallace
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Bacterial Communities ◽  
Peptide Nucleic Acid ◽  
Pcr Amplification ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Methods Development ◽  
Significant Bias ◽  
Plant Organelles

AbstractPlant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16s rRNA gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially since the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16s rRNA amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct “fingerprint” of its own biases. Plant samples were less clear, with different species having different “best” options. For 16s amplification, we find that using peptide nucleic acid (PNA) clamps provides the least taxonomic distortion, while chloroplast-discriminating primers are easy and inexpensive but present significant bias in the results. We do not recommend blocking oligos, as they involved a more complex protocol and showed significant taxonomic bias in the results. Further methods development will hopefully result in protocols that are even more reliable and less biased.

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