Preparation of Fungal ITS Amplicons from Soil Samples for Illumina Amplicon Sequencing v1 (protocols.io.nmgdc3w)

protocols.io ◽  
2018 ◽  
Author(s):  
Loreen Sommermann ◽  
Joerg Geistlinger ◽  
Jessica Zwanzig ◽  
Ingo Schellenberg
Keyword(s):  
Amplicon Sequencing ◽  
Soil Samples ◽  
Fungal Its ◽  
Illumina Amplicon Sequencing

scholarly journals A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1274
Author(s):  
Ko-Hsuan Chen ◽  
Reid Longley ◽  
Gregory Bonito ◽  
Hui-Ling Liao
Keyword(s):  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Agricultural Systems ◽  
Specific Primers ◽  
Community Discovery ◽  
16S Ribosomal Dna ◽  
Genomic Regions ◽  
Internal Transcribed Spacer Rdna ◽  
Fungal Its ◽  
Illumina Amplicon Sequencing

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

scholarly journals Identifying Hidden Viable Bacterial Taxa in Tropical Forest Soils Using Amplicon Sequencing of Enrichment Cultures

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 569
Author(s):  
Chakriya Sansupa ◽  
Sara Fareed Mohamed Wahdan ◽  
Terd Disayathanoowat ◽  
Witoon Purahong
Keyword(s):  
Bacterial Community ◽  
Forest Soil ◽  
Culture Media ◽  
Enrichment Culture ◽  
Sequence Similarity ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Enrichment Cultures ◽  
Total Community

This study aims to estimate the proportion and diversity of soil bacteria derived from eDNA-based and culture-based methods. Specifically, we used Illumina Miseq to sequence and characterize the bacterial communities from (i) DNA extracted directly from forest soil and (ii) DNA extracted from a mixture of bacterial colonies obtained by enrichment cultures on agar plates of the same forest soil samples. The amplicon sequencing of enrichment cultures allowed us to rapidly screen a culturable community in an environmental sample. In comparison with an eDNA community (based on a 97% sequence similarity threshold), the fact that enrichment cultures could capture both rare and abundant bacterial taxa in forest soil samples was demonstrated. Enrichment culture and eDNA communities shared 2% of OTUs detected in total community, whereas 88% of enrichment cultures community (15% of total community) could not be detected by eDNA. The enrichment culture-based methods observed 17% of the bacteria in total community. FAPROTAX functional prediction showed that the rare and unique taxa, which were detected with the enrichment cultures, have potential to perform important functions in soil systems. We suggest that enrichment culture-based amplicon sequencing could be a beneficial approach to evaluate a cultured bacterial community. Combining this approach together with the eDNA method could provide more comprehensive information of a bacterial community. We expected that more unique cultured taxa could be detected if further studies used both selective and non-selective culture media to enrich bacteria at the first step.

scholarly journals Inoculation with Rhizophagus irregularis Does Not Alter Arbuscular Mycorrhizal Fungal Community Structure within the Roots of Corn, Wheat, and Soybean Crops

2020 ◽  
Vol 8 (1) ◽  
pp. 83 ◽  
Author(s):  
Sébastien Renaut ◽  
Rachid Daoud ◽  
Jacynthe Masse ◽  
Agathe Vialle ◽  
Mohamed Hijri
Keyword(s):  
Community Composition ◽  
Arbuscular Mycorrhizal ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Rhizophagus Irregularis ◽  
Community Diversity ◽  
Reference Sequence ◽  
Growth Stages ◽  
Arbuscular Mycorrhizal Fungal

Little is known about establishment success of the arbuscular mycorrhizal fungal (AMF) inocula and their effects on a soil-indigenous community of AMF. In this study, we assessed the effect of introducing Rhizophagus irregularis DAOM-197198 in soil under field condition on the community composition of indigenous AMF in the roots of corn (Zea mays), soybean (Glycine max), and wheat (Triticum aestivum). Three field trials were conducted with inoculated and non-inoculated plots. Four to ten roots and their rhizosphere soil samples of two growth stages for corn and wheat, and one growing stage of soybean, were collected, totalling 122 root and soil samples. Root colonization was measured microscopically, and the fungal communities were determined by paired-end Illumina MiSeq amplicon sequencing using 18S rDNA marker. After quality trimming and merging of paired ends, 6.7 million sequences could be assigned to 414 different operational taxonomic units. These could be assigned to 68 virtual taxa (VT) using the AMF reference sequence database MaarjAM. The most abundant VT corresponded to R. irregularis. The inoculation treatment did not influence the presence of R. irregularis, or AMF community diversity in roots. This seems to indicate that inoculation with R. irregularis DAOM-197198 does not change the indigenous AMF community composition, probably because it is already present in high abundance naturally.

scholarly journals Homogenization of Rhizosphere Bacterial Communities by Pea (Pisum sativum L.) Cultivated under Different Conservation Agricultural Practices in the Eastern Himalayas

2019 ◽  
Author(s):  
Diptaraj Chaudhari ◽  
Krishnappa Rangappa ◽  
Anup Das ◽  
Jayanta Layek ◽  
Savita Basavaraju ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Management Practices ◽  
Soil Health ◽  
Agricultural Practices ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Residue Management ◽  
Bulk Soil ◽  
Rrna Gene ◽  
North East

AbstractConservation agriculture offers a suitable system to harmonize agriculture with the environment, especially in fragile ecosystems of North-East India. Soil microbes play pivotal roles in ecosystem functioning and act as indispensable indicators of overall fitness of crop plant and soil health. Here we demonstrated that altercations in residue management and tillage practices lead to the development of differential bacterial communities forcing the pea plants to recruit special groups of bacteria leading to highly homogenous rhizosphere communities. Pea rhizosphere and bulk soil samples were collected, and bacterial community structure was estimated by 16S rRNA gene amplicon sequencing and predictive functional analysis was performed using Tax4Fun. The effect on pea plants was evident in the bacterial communities as the overall diversity of rhizosphere samples was significantly higher to that of bulk soil samples. Bacillus, Staphylococcus, Planomicrobium, Enterobacter, Arthrobacter, Nitrobacter, Geobacter, and Sphingomonas were noticed as the most abundant genera in the rhizosphere and bulk soil samples. The abundance of Firmicutes and Proteobacteria altered significantly in the rhizosphere and bulk samples, which was further validated by qPCR. Selection of specific taxa by pea plant was indicated by the higher values of mean proportion of Rhizobium, Pseudomonas, Pantoea, Nitrobacter, Enterobacter and Sphingomonas in rhizosphere samples, and Massilia, Paenibacillus and Planomicrobium in bulk soil samples. Tillage and residue management treatments did not significantly alter the bacterial diversity, while their influence was observed on the abundance of few genera. Recorded results revealed that pea plant selects specific taxa into its rhizosphere plausibly to meet its requirements for nutrient uptake and stress amelioration under the different tillage and residue management practices.

Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

2020 ◽  
Author(s):  
Yingwu Shi ◽  
Hongmei Yang ◽  
Min Chu ◽  
Xinxiang Niu ◽  
Xiangdong Huo ◽  
...  
Keyword(s):  
Community Structure ◽  
Endophytic Bacteria ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Operational Taxonomic Units ◽  
Cotton Sample ◽  
Endophytic Bacterial Community ◽  
V3 Region ◽  
Illumina Amplicon Sequencing

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

Alteration of crop rotation in continuous Pinellia ternate cropping soils profiled via fungal ITS amplicon sequencing

2019 ◽  
Vol 68 (6) ◽  
pp. 522-529 ◽  
Author(s):  
Z. He ◽  
H. Chen ◽  
L. Liang ◽  
J. Dong ◽  
Z. Liang ◽  
...  
Keyword(s):  
Crop Rotation ◽  
Amplicon Sequencing ◽  
Fungal Its

scholarly journals Limited added value of fungal ITS amplicon sequencing in the study of bovine abortion

Heliyon ◽  
2018 ◽  
Vol 4 (11) ◽  
pp. e00915 ◽  
Author(s):  
Sara Vidal ◽  
Bernd W. Brandt ◽  
Martina Dettwiler ◽  
Carlos Abril ◽  
Jenny Bressan ◽  
...  
Keyword(s):  
Amplicon Sequencing ◽  
Added Value ◽  
Bovine Abortion ◽  
Fungal Its

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.

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