<scp>DNA</scp>
            extraction from benthic Cyanobacteria: comparative assessment and optimization

Gaget, Virginie; Keulen, Angela; Lau, Melody; Monis, Paul; Brookes, Justin D.

doi:10.1111/jam.13332

Cited by 20 publications

(22 citation statements)

References 29 publications

(45 reference statements)

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“…Therefore, ne needs to be careful when using buffers that contain ETDA since this can be found commonly in some elution buffers of commercial kits and in some certain concentrations it may deplete magnesium ions and thus inhibit DNA polymerase activity (Schrader et al 2012). Also, commercial kits are expensive when a large number of samples need to be processed, while the lab made buffer for DNA extraction represent a more economic extraction method if the efficiency criteria is required.Within this study we tested both several commercial kits and a lab made buffer for the cyanobacteria extraction since it was proven that the extracted DNA concentration varied significantly between the commercial kits (Gaget et al 2017), which is also in line with our findings (Table 3, test ).…”

Section: Different Matrix and Dna Extraction Kitssupporting

confidence: 86%

“…Testing the DNA extraction methods is an important and mandatory step no matter what the matrix is, as previous studies showed (Jiang et al, 2005;Gaget et al, 2017), since this represent a critical step in culture-independent bacterial profiling (Hallmaier-Wacker et al 2018). Even if many different protocols and commercially kits have been developed for DNA extractions from environmental samples (Chandraa et al 2010) there is still a lack of reliable techniques for DNA extraction as well for RNA isolation for several types of cyanobacteria (Tiam et al 2019).…”

Section: Different Matrix and Dna Extraction Kitsmentioning

confidence: 99%

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

Postolache

2021

Preprint

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Molecular 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.

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Section: Different Matrix and Dna Extraction Kitssupporting

confidence: 86%

Section: Different Matrix and Dna Extraction Kitsmentioning

confidence: 99%

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

Postolache

2021

Preprint

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“…While DNA extraction method and primer choice can be sources of bias in 16S rRNA gene‐based analyses (Feinstein, Sul, & Blackwood, ; Tremblay et al., ), we believe these are unlikely to be the cause of the relatively low abundance of cyanobacteria in our samples. While calcified cyanobacterial sheaths were abundant in LB microbialites, previous studies of similar systems (e.g., stromatolites, hot springs) found DNA extraction kits that include a physical disruption step (such as the MoBio PowerSoil kit used here) were quite effective at lysing cyanobacterial cell walls (Balskus, Case, & Walsh, ; Gaget, Keulen, Lau, Monis, & Brookes, ; Pepe‐Ranney et al., ). Primer bias against cyanobacteria or specific cyanobacterial classes is also unlikely, as a systematic evaluation of the 515F‐806R primer pair (Bates et al., ) using SILVA's TestPrime tool (Klindworth et al., ) found that these primers matched >87% of existing 16S rRNA gene sequences classified as part of the phylum cyanobacteria in the SILVA SSU Ref NR database (Version 128; Quast et al., ).…”

Section: Discussionmentioning

confidence: 75%

“…While calcified cyanobacterial sheaths were abundant in LB microbialites, previous studies of similar systems (e.g., stromatolites, hot springs) found DNA extraction kits that include a physical disruption step (such as the MoBio PowerSoil kit used here) were quite effective at lysing cyanobacterial cell walls (Balskus, Case, & Walsh, 2011;Gaget, Keulen, Lau, Monis, & Brookes, 2017;Pepe-Ranney et al, 2012). Primer bias against cyanobacteria or specific cyanobacterial classes is also unlikely, as a systematic evaluation of the 515F-806R primer pair (Bates et al, 2011) using SILVA's TestPrime tool (Klindworth et al, 2013) found that these primers matched ites in other locations (Allen et al, 2010;Dupraz & Visscher, 2005).…”

Section: Mramentioning

confidence: 99%

Microbial diversity and biomarker analysis of modern freshwater microbialites from Laguna Bacalar, Mexico

et al. 2018

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Laguna Bacalar is a sulfate-rich freshwater lake on the Yucatan Peninsula that hosts large microbialites. High sulfate concentrations distinguish Laguna Bacalar from other freshwater microbialite sites such as Pavilion Lake and Alchichica, Mexico, as well as from other aqueous features on the Yucatan Peninsula. While cyanobacterial populations have been described here previously, this study offers a more complete characterization of the microbial populations and corresponding biogeochemical cycling using a three-pronged geobiological approach of microscopy, high-throughput DNA sequencing, and lipid biomarker analyses. We identify and compare diverse microbial communities of Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria that vary with location along a bank-to-bank transect across the lake, within microbialites, and within a neighboring mangrove root agglomeration. In particular, sulfate-reducing bacteria are extremely common and diverse, constituting 7%-19% of phylogenetic diversity within the microbialites, and are hypothesized to significantly influence carbonate precipitation. In contrast, Cyanobacteria account for less than 1% of phylogenetic diversity. The distribution of lipid biomarkers reflects these changes in microbial ecology, providing meaningful biosignatures for the microbes in this system. Polysaturated short-chain fatty acids characteristic of cyanobacteria account for <3% of total abundance in Laguna Bacalar microbialites. By contrast, even short-chain and monounsaturated short-chain fatty acids attributable to both Cyanobacteria and many other organisms including types of Alphaproteobacteria and Gammaproteobacteria constitute 43%-69% and 17%-25%, respectively, of total abundance in microbialites. While cyanobacteria are the largest and most visible microbes within these microbialites and dominate the mangrove root agglomeration, it is clear that their smaller, metabolically diverse associates are responsible for significant biogeochemical cycling in this microbialite system.

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“…Molecular methods also have weaknesses of their own. For instance, the DNA extraction step is an important source of bias in molecular investigations of cyanobacterial assemblages, as high amounts of EPS in the biofilm matrix interfere considerably with cell lysis procedures [54]. Moreover, using data from artificial communities, Pessi et al [26] showed that PCR and sequencing biases shift considerably the observed relative abundances of individual cyanobacterial taxa.…”

Section: Discussionmentioning

confidence: 99%

Marked Succession of Cyanobacterial Communities Following Glacier Retreat in the High Arctic

et al. 2018

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Cyanobacteria are important colonizers of recently deglaciated proglacial soil but an in-depth investigation of cyanobacterial succession following glacier retreat has not yet been carried out. Here, we report on the successional trajectories of cyanobacterial communities in biological soil crusts (BSCs) along a 100-year deglaciation gradient in three glacier forefields in central Svalbard, High Arctic. Distance from the glacier terminus was used as a proxy for soil age (years since deglaciation), and cyanobacterial abundance and community composition were evaluated by epifluorescence microscopy and pyrosequencing of partial 16S rRNA gene sequences, respectively. Succession was characterized by a decrease in phylotype richness and a marked shift in community structure, resulting in a clear separation between early (10-20 years since deglaciation), mid (30-50 years), and late (80-100 years) communities. Changes in cyanobacterial community structure were mainly connected with soil age and associated shifts in soil chemical composition (mainly moisture, SOC, SMN, K, and Na concentrations). Phylotypes associated with early communities were related either to potentially novel lineages (< 97.5% similar to sequences currently available in GenBank) or lineages predominantly restricted to polar and alpine biotopes, suggesting that the initial colonization of proglacial soil is accomplished by cyanobacteria transported from nearby glacial environments. Late communities, on the other hand, included more widely distributed genotypes, which appear to establish only after the microenvironment has been modified by the pioneering taxa.

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DNA extraction from benthic Cyanobacteria: comparative assessment and optimization

Cited by 20 publications

References 29 publications

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

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

Microbial diversity and biomarker analysis of modern freshwater microbialites from Laguna Bacalar, Mexico

Marked Succession of Cyanobacterial Communities Following Glacier Retreat in the High Arctic

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