Addressing PCR Biases in Environmental Microbiology Studies

Sipos, Rita; Székely, Anna J.; Révész, Sára; Màrialigeti, Károly

doi:10.1007/978-1-60761-439-5_3

Cited by 32 publications

(24 citation statements)

References 60 publications

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“…Efforts to focus on unculturable microbes resulted in development of various “universal” primers that targeted ribosomal genes of diverse microbial taxa [50], [51], [52]. These primers reveal diverse microbes undetectable by isolation based techniques, but may fail to selectively amplify only fungal sequences from mixed samples, or to amplify diverse fungi with equal efficiencys [42], [53], [54].…”

Section: Discussionmentioning

confidence: 99%

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

et al. 2011

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Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities.

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Section: Discussionmentioning

confidence: 99%

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

et al. 2011

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“…However, the data generated need to be interpreted with the awareness of culture‐independent PCR biases that have been reviewed elsewhere (Sipos et al ., 2010), such as the possibility of preferential amplification, due to the different efficiency of the primer towards selected species, that may result in the under‐representation of some clades (Pinto and Raskin, 2012). …”

Section: Monitoring Microbes In Food Fermentationsmentioning

confidence: 99%

Metagenomics insights into food fermentations

Filippis

Parente

2016

Microbial Biotechnology

197

117

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SummaryThis review describes the recent advances in the study of food microbial ecology, with a focus on food fermentations. High‐throughput sequencing (HTS) technologies have been widely applied to the study of food microbial consortia and the different applications of HTS technologies were exploited in order to monitor microbial dynamics in food fermentative processes. Phylobiomics was the most explored application in the past decade. Metagenomics and metatranscriptomics, although still underexploited, promise to uncover the functionality of complex microbial consortia. The new knowledge acquired will help to understand how to make a profitable use of microbial genetic resources and modulate key activities of beneficial microbes in order to ensure process efficiency, product quality and safety.

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“…PCR amplification for each ATAD DNA extract was also carried out using universal bacterial 1492 r and 27 f primers (Table 1) via touchdown PCR, and low cycle number [62][63][64] to avoid PCR biases. PCR products were ligated into the pGEM-TA vector (Promega), transformed into electrocomptent JM109 cells and positive clones with vector insert of correct size confirmed by amplification with vector specific primers T7 f and SP6 r (Table 1).…”

Section: Analysis Of the Total Bacterial Community Profile And Predommentioning

confidence: 99%

Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery

2010

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Molecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments. OPEN ACCESSDiversity 2010, 2 506

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Addressing PCR Biases in Environmental Microbiology Studies

Cited by 32 publications

References 60 publications

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

Metagenomics insights into food fermentations

Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery

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