scholarly journals Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits

2021 ◽  
Author(s):  
Kathrin Janssen ◽  
Shook Ling Low ◽  
Yan Wang ◽  
Qi‐Yong Mu ◽  
Gabriele Bierbaum ◽  
...  
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Water Lily

scholarly journals Optimized Protocol for Cyanobacterial 16S rRNA Analysis in Danube Delta Lakes

2021 ◽  
Author(s):  
Maria Iasmina Moza ◽  
Carmen Postolache
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Phytoplankton Biomass ◽  
Extraction Method ◽  
Seasonal Difference ◽  
Danube Delta ◽  
16S Rrna Analysis ◽  
Dna Extraction Method ◽  
Optimized Protocol ◽  
Cyanobacterial Biomass

AbstractMolecular biology protocols have been more and more accessible to researchers for ecological investigations, however, these protocols always require optimization steps for the analysis of specific types of samples. The purpose of this study was to optimize a molecular protocol for the analysis of cyanobacterial 16S rRNA in Danube Delta shallows lakes. In this regard, several commercial DNA extraction kits were tested in comparison with potassium ethyl xanthogenate extraction method on different matrices. The obtained DNA was further used for 16S rRNA PCR optimization. Finally, an optimized protocol is proposed for the molecular analysis of cyanobacteria group in freshwater samples. The best DNA extraction method was the potassium xanthogenate extraction from dried cyanobacterial biomass. A dynamic in total genomic eDNA was observed, reflecting the seasonal difference in phytoplankton biomass from the studied lakes. The PCR protocol optimized by us can be successfully applied for the identification of a broad range of cyanobacterial genetic markers.

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

1992 ◽  
Vol 100 (1-3) ◽  
pp. 59-65 ◽  
Author(s):  
Paul A. Rochelle ◽  
John C. Fry ◽  
R. John Parkes ◽  
Andrew J. Weightman
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Deep Sediment

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

scholarly journals The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Ingo C. Starke ◽  
Wilfried Vahjen ◽  
Robert Pieper ◽  
Jürgen Zentek
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Dna Extraction ◽  
16S Rrna Genes ◽  
Read Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Extraction Procedures ◽  
Primer Sets

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

scholarly journals Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

2020 ◽  
Author(s):  
CARMEN HARO ◽  
MANUEL ANGUITA-MAESO ◽  
Madis Metsis ◽  
JUAN A NAVAS-CORTES ◽  
BLANCA LANDA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Bacterial Communities ◽  
Methodological Approach ◽  
Xylem Sap ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Community Assessment ◽  
Woody Crop

Next Generation Sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a nonbiased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from 'Picual' and 'Arbequina' olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UNIFRAC distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction Kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region and the Silva 132 database for taxonomic assignation. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting within the xylem sap of olives or other woody crop species.

Genetic Diversity of the Biofilm CoveringMontacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

1998 ◽  
Vol 64 (9) ◽  
pp. 3464-3472 ◽  
Author(s):  
David C. Gillan ◽  
Arjen G. C. L. Speksnijder ◽  
Gabriel Zwart ◽  
Chantal De Ridder
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Dna Extraction ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis ◽  
Biofilm Bacteria ◽  
Sequence Types

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas,Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of theCytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification

2015 ◽  
Vol 99 (20) ◽  
pp. 8731-8740 ◽  
Author(s):  
Qi Yang ◽  
Christopher M. M. Franco ◽  
Wei Zhang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Gene Amplification ◽  
Rrna Gene

scholarly journals Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

2014 ◽  
Vol 80 (6) ◽  
pp. 1985-1994 ◽  
Author(s):  
Yuki Morono ◽  
Takeshi Terada ◽  
Tatsuhiko Hoshino ◽  
Fumio Inagaki
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Dna Extraction ◽  
Extraction Method ◽  
Cell Disruption ◽  
Rrna Genes ◽  
Dna Integrity ◽  
Microbial Cells ◽  
Sediment Samples ◽  
Dna Extraction Method

ABSTRACTA prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.

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