Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region

Hansen, Michael R.; Tolker‐Nielsen, Tim; Givskov, Michael; Molin, Soeren

doi:10.1111/j.1574-6941.1998.tb00500.x

Cited by 197 publications

(102 citation statements)

References 18 publications

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“…PCR amplification of the V3 region of the 16S rRNA gene The total DNA in replicate samples was pooled together for PCR using the forward primer U341F (5 0 -CCTACGGGRSGCAGCAG-3 0 ) (Hansen et al, 1998) and reverse primer R685 (5 0 -ATCTACGC ATTTCACCGCTAC-3 0 ) for bacteria (Wang et al, 2004), and the forward primer A344F (5 0 -AYGGGGY GCASCAGGSG-3 0 ) and reverse primer A519R (5 0 -GG TDTTACCGCGGCKGCTG-3 0 ) for archaea (Teske and Sorensen, 2007) in separate reactions. A set of sixnucleotide (nt) barcodes was designed and added to the 5 0 end of U341F, R685, A344F and A519R for the multiplexing of samples in the pyrosequencing runs.…”

Section: Methodsmentioning

confidence: 99%

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

et al. 2010

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The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea.

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Section: Methodsmentioning

confidence: 99%

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

et al. 2010

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“…with the Qiagen kit suggests that the bias was not introduced at the DNAextraction phase. Differences in secondary structure affecting either the availability of the priming sites or the polymerization reaction may cause amplification bias [11]. In any case, our results emphasize that either multiple primer pairs or both molecular and culture-based approaches should be used when characterizing microbial communities [11,26].…”

Section: MD Shawkey Et Al: Microbial Diversity Of Bird Feathersmentioning

confidence: 94%

Microbial Diversity of Wild Bird Feathers Revealed throughCulture-Based and Culture-Independent Techniques

et al. 2005

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Despite recent interest in the interactions between birds and environmental microbes, the identities of the bacteria that inhabit the feathers of wild birds remain largely unknown. We used culture-based and culture-independent surveys of the feathers of eastern bluebirds (Sialis sialis) to examine bacterial flora. When used to analyze feathers taken from the same birds, the two survey techniques produced different results. Species of the poorly defined genus Pseudomonas were most common in the molecular survey, whereas species of the genus Bacillus were predominant in the culture-based survey. This difference may have been caused by biases in both the culture and polymerase chain reaction techniques that we used. The pooled results from both techniques indicate that the overall community is diverse and composed largely of members of the Firmicutes and b-and c-subdivisions of the Proteobacteria. For the most part, bacterial sequences isolated from birds were closely related to sequences of soil-borne and water-borne bacteria in the GenBank database, suggesting that birds may have acquired many of these bacteria from the environment. However, the metabolic properties and optimal growth requirements of several isolates suggest that some of the bacteria may have a specialized association with feathers.

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“…Bias in phylogenetic analysis is introduced through differential amplification caused by differences in the efficiency of primer binding, interference by sequences flanking primer regions (Hansen et al, 1998) and differences in the kinetics of the PCR reaction. (e.g., Brunk and Eis, 1998;Reysenbach and Pace, 1995;Suzuki and Giovanni, 1996).…”

Section: Discussionmentioning

confidence: 99%

Review and re-analysis of domain-specific 16S primers

Baker

Smith

Cowan

2003

Journal of Microbiological Methods

1,620

1,071

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The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the ''universal'' primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective 'universal' Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.

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Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region

Cited by 197 publications

References 18 publications

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

Microbial Diversity of Wild Bird Feathers Revealed throughCulture-Based and Culture-Independent Techniques

Review and re-analysis of domain-specific 16S primers

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