scholarly journals A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform

2017 ◽  
Author(s):  
Monica Pichler ◽  
Ömer K. Coskun ◽  
Ana Sofia Ortega ◽  
Nicola Conci ◽  
Gert Wörheide ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Environmental Samples ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Mock Community ◽  
Rrna Gene Sequencing

ABSTRACTHigh-throughput sequencing of the 16S rRNA gene is widely used in microbial ecology, with Illumina platforms being widely used in recent studies. The MiniSeq, Illumina’s latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger sequencing platforms (e.g. MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed an additional sequencing primer that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples as a quality control benchmark. After careful filtering procedures, we were able to recapture a realistic richness of the mock community, and identify meaningful differences in alpha and beta diversity in the environmental samples. These results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.IMPORTANCEWe modified a custom sequencing approach and used a mock community to test the fidelity of high-throughput sequencing on the Illumina MiniSeq platform. Our results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq. In addition, our protocol increases feasibility for small laboratories to perform their own high-throughput sequencing of the 16S rRNA marker gene.

scholarly journals High-Throughput Sequencing for Examining Salmonella Prevalence and Pathogen—Microbiota Relationships in Barn Swallows

2021 ◽  
Vol 9 ◽  
Author(s):  
Olivia N. Choi ◽  
Ammon Corl ◽  
Andrew Wolfenden ◽  
Avishai Lublin ◽  
Suzanne L. Ishaq ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Barn Swallows ◽  
Rrna Gene Sequencing

Studies in both humans and model organisms suggest that the microbiome may play a significant role in host health, including digestion and immune function. Microbiota can offer protection from exogenous pathogens through colonization resistance, but microbial dysbiosis in the gastrointestinal tract can decrease resistance and is associated with pathogenesis. Little is known about the effects of potential pathogens, such as Salmonella, on the microbiome in wildlife, which are known to play an important role in disease transmission to humans. Culturing techniques have traditionally been used to detect pathogens, but recent studies have utilized high throughput sequencing of the 16S rRNA gene to characterize host-associated microbial communities (i.e., the microbiome) and to detect specific bacteria. Building upon this work, we evaluated the utility of high throughput 16S rRNA gene sequencing for potential bacterial pathogen detection in barn swallows (Hirundo rustica) and used these data to explore relationships between potential pathogens and microbiota. To accomplish this, we first compared the detection of Salmonella spp. in swallows using 16S rRNA data with standard culture techniques. Second, we examined the prevalence of Salmonella using 16S rRNA data and examined the relationship between Salmonella-presence or -absence and individual host factors. Lastly, we evaluated host-associated bacterial diversity and community composition in Salmonella-present vs. -absent birds. Out of 108 samples, we detected Salmonella in six (5.6%) samples based on culture, 25 (23.1%) samples with unrarefied 16S rRNA gene sequencing data, and three (2.8%) samples with both techniques. We found that sex, migratory status, and weight were correlated with Salmonella presence in swallows. In addition, bacterial community composition and diversity differed between birds based on Salmonella status. This study highlights the value of 16S rRNA gene sequencing data for monitoring pathogens in wild birds and investigating the ecology of host microbe-pathogen relationships, data which are important for prediction and mitigation of disease spillover into domestic animals and humans.

scholarly journals Bacterial Communities in Serpa Cheese by Culture Dependent Techniques, 16S rRNA Gene Sequencing and High-throughput Sequencing Analysis

2018 ◽  
Vol 83 (5) ◽  
pp. 1333-1341 ◽  
Author(s):  
Maria Teresa P. Gonçalves ◽  
María José Benito ◽  
María de Guía Córdoba ◽  
Conceição Egas ◽  
Almudena V. Merchán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Communities ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Rrna Gene Sequencing ◽  
Culture Dependent

scholarly journals High-throughput 16S rRNA gene sequencing reveals that 6-hydroxydopamine affects gut microbial environment

PLoS ONE ◽  
2019 ◽  
Vol 14 (8) ◽  
pp. e0217194 ◽  
Author(s):  
Jin Gyu Choi ◽  
Eugene Huh ◽  
Namkwon Kim ◽  
Dong-Hyun Kim ◽  
Myung Sook Oh
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Microbial Environment ◽  
6 Hydroxydopamine

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

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