Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases

Hugenholtz, Philip; Huber, Thomas

doi:10.1099/ijs.0.02441-0

Cited by 206 publications

(168 citation statements)

References 25 publications

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“…Novel DNA-sequencing methods using shorter DNA read length should result in even fewer chimeric junctions per read as demonstrated for single cells by the 454 Life Sciences method (Marcy et al, 2007a). Nevertheless, the appearance of MDA-associated chimeras in databases of environmental DNA sequences is a concern, as has already occurred for the PCR method in the case of ribosomal sequence databases (Hugenholtz and Huber, 2003;Ashelford et al, 2005). It will be important to reduce the formation of chimeras further, and identifying their cause (Lasken and Stockwell, 2007) is a substantial step in this direction.…”

Section: Resultsmentioning

confidence: 99%

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

2008

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Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

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Section: Resultsmentioning

confidence: 99%

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

2008

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“…D1-D2 LSU rDNA fragments show virtually no intraspecific variations while discriminating morphospecies that split in the Pleistocene. Unambiguous LSU rDNA sequences were first screened for chimeras using Check-Chimera (36), then double checked by thorough visual inspection of all sequences producing abnormally long branches in neighbor-joining trees (37) as described in Hugenholtz and Huber (38). This conservative approach led to the removal of Ϸ13% of putative chimeric sequences from subsequent analyses.…”

Section: Methodsmentioning

confidence: 99%

Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans

Hui

Probert

Uitz

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

266

190

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The current paradigm holds that cyanobacteria, which evolved oxygenic photosynthesis more than 2 billion years ago, are still the major light harvesters driving primary productivity in open oceans. Here we show that tiny unicellular eukaryotes belonging to the photosynthetic lineage of the Haptophyta are dramatically diverse and ecologically dominant in the planktonic photic realm. The use of Haptophyta-specific primers and PCR conditions adapted for GC-rich genomes circumvented biases inherent in classical genetic approaches to exploring environmental eukaryotic biodiversity and led to the discovery of hundreds of unique haptophyte taxa in 5 clone libraries from subpolar and subtropical oceanic waters. Phylogenetic analyses suggest that this diversity emerged in Paleozoic oceans, thrived and diversified in the permanently oxygenated Mesozoic Panthalassa, and currently comprises thousands of ribotypic species, belonging primarily to low-abundance and ancient lineages of the ''rare biosphere.'' This extreme biodiversity coincides with the pervasive presence in the photic zone of the world ocean of 19 -hexanoyloxyfucoxanthin (19-Hex), an accessory photosynthetic pigment found exclusively in chloroplasts of haptophyte origin. Our new estimates of depth-integrated relative abundance of 19-Hex indicate that haptophytes dominate the chlorophyll a-normalized phytoplankton standing stock in modern oceans. Their ecologic and evolutionary success, arguably based on mixotrophy, may have significantly impacted the oceanic carbon pump. These results add to the growing evidence that the evolution of complex microbial eukaryotic cells is a critical force in the functioning of the biosphere.Haptophyta ͉ photosynthesis ͉ protistan biodiversity ͉ eukaryotic biodiversity O xygenic photosynthesis, the most complex and energetically powerful molecular process in biology, originated in cyanobacteria more than 2 billion years ago in Archean oceans (1). Marine photosynthesis still contributes Ϸ50% of total primary production on Earth (2). This revolutionary process was integrated, at least once, into an ancestral phagotrophic eukaryotic lineage through the evolution of chloroplasts, which themselves were redistributed to a large variety of aquatic eukaryote lineages via permanent secondary and tertiary endosymbioses (3). Despite this evolutionary trend from photosynthetic prokaryotes to eukaryotes, particularly visible in today's coastal oceans where microalgae such as diatoms and dinoflagellates are omnipresent, cyanobacteria have been repeatedly claimed as the champions of photosynthesis in open ocean waters (4). This hypothesis followed the introduction of flow cytometry and molecular genetic approaches to biological oceanography in the 1980s, which revealed astonishing concentrations of minute cyanobacterial cells of the genera Procholorococcus and Synechococcus in marine waters (5). The physiology, ecology, and functional and environmental genomics of these prokaryotes are subjects of ongoing intensive study (6).Several lines of e...

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“…The sequences were edited using Sequencer DNA sequencing software (Gene Codes Inc.). Checks for chimeric sequences were conducted using the CHECK_CHIMERA program from the Ribosomal Database Project (http://rdp.cme.msu.edu) and the program BELLEROPHON (Hugenholtz & Huber, 2003). 16S rRNA gene sequences of selected relatives within the phylum Bacteroidetes were compiled and aligned using the automatic nucleic acid aligner in the ARB software package (Ludwig et al, 2004), and alignments were refined manually.…”

Section: Methodsmentioning

confidence: 99%

Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants

Kragelund

Levantesi

Borger³

et al. 2008

Microbiology

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Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.

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Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases

Cited by 206 publications

References 25 publications

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans

Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants

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