Structure, diversity and potential metabolic activity of prokaryotic communities of Ebro Delta wetlands (Spain) as revealed by 16S rRNA gene amplicon sequencing

The monitoring of the structure and potential function of the prokaryotic assemblages of aquatic ecosystems can be used for the assessment of ecosystem health. In this work, we analysed by 16S rRNA gene amplicon massive sequencing the aquatic and sediment prokaryotic communities inhabiting three wetlands located in Ebro river Delta (Spain), which differ in their salinity and trophic status. We extracted the DNA from both matrices and sequenced the V4 region of the prokaryotic 16S rRNA gene by Illumina sequencing. The raw sequences obtained were processed and clustered in ZOTUs, which are sequences at 100% identity that were the basis for the structural and statistical analyses. Our results showed that water communities have less diversity and evenness than sediment communities. Moreover, statistical analyses showed that salinity is the main environmental factor that affects the structuration of sediment prokaryotic communities, while seasonality is also a very additional important environmental driver for aquatic communities. We also studied the relationship between the environmental factors and the potential metabolism of the aquatic and sediment prokaryotic communities, specifically for the main metabolic processes of the C-cycle. Focusing on sediment, we observed that methanogenic archaea are present not only in freshwater environments but also in the saline wetlands, though the observed methane emissions decreased with salinity. To explain this, we studied the potential activity of sulfate-reducing bacteria (SRB) and methanogenic archaea inferring the expression of genes dsrB and mcrA, which are gene markers for dissimilatory sulfate reduction and methanogenesis, respectively. We observed that, in saline wetlands, the potential activity of methanogens decreases because SRB bacteria outcompete them due to the higher sulfate availability enhancing sulfate-reduction. In the sediment communities of low salinity wetlands the relevance of methanogens is lower in terms of relative abundance, but the methane emissions are higher due to the lower sulfate availability. On the other hand, aquatic methanotrophic bacteria are very important in the net balance of methane emissions in Ebro Delta wetlands, as their relative abundance in the water column influences the amount of methane that is finally released to the atmosphere. In conclusion, our study deepens in the relationship between prokaryotic community structure and function in deltaic wetlands, giving a comprehensive overview of factors influencing the ecological health and certain processes, such as methane emissions, in deltaic systems.

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Soil Depth Determines the Composition and Diversity of Bacterial and Archaeal Communities in a Poplar Plantation

Forests ◽

10.3390/f10070550 ◽

2019 ◽

Vol 10 (7) ◽

pp. 550 ◽

Cited By ~ 6

Author(s):

Huili Feng ◽

Jiahuan Guo ◽

Weifeng Wang ◽

Xinzhang Song ◽

Shuiqiang Yu

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Relative Abundance ◽

Rrna Gene ◽

Poplar Plantation ◽

Plantation Forest ◽

Bacterial And Archaeal Communities ◽

Different Depths ◽

Archaeal Communities

Understanding the composition and diversity of soil microorganisms that typically mediate the soil biogeochemical cycle is crucial for estimating greenhouse gas flux and mitigating global changes in plantation forests. Therefore, the objectives of this study were to investigate changes in diversity and relative abundance of bacteria and archaea with soil profiles and the potential factors influencing the vertical differentiation of microbial communities in a poplar plantation. We investigated soil bacterial and archaeal community compositions and diversities by 16S rRNA gene Illumina MiSeq sequencing at different depths of a poplar plantation forest in Chenwei forest farm, Sihong County, Jiangsu, China. More than 882,422 quality-filtered 16S rRNA gene sequences were obtained from 15 samples, corresponding to 34 classified phyla and 68 known classes. Ten major bacterial phyla and two archaeal phyla were found. The diversity of bacterial and archaeal communities decreased with depth of the plantation soil. Analysis of variance (ANOVA) of relative abundance of microbial communities exhibited that Nitrospirae, Verrucomicrobia, Latescibacteria, GAL15, SBR1093, and Euryarchaeota had significant differences at different depths. The transition zone of the community composition between the surface and subsurface occurred at 10–20 cm. Overall, our findings highlighted the importance of depth with regard to the complexity and diversity of microbial community composition in plantation forest soils.

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Impact of a probiotic, inulin, or their combination on the piglets’ microbiota at different intestinal locations

Beneficial Microbes ◽

10.3920/bm2014.0030 ◽

2015 ◽

Vol 6 (4) ◽

pp. 473-483 ◽

Cited By ~ 10

Author(s):

V.A. Sattler ◽

K. Bayer ◽

G. Schatzmayr ◽

A.G. Haslberger ◽

V. Klose

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Real Time ◽

Relative Abundance ◽

Real Time Pcr ◽

Denaturing Gradient Gel Electrophoresis ◽

Feed Additives ◽

Rrna Gene ◽

16S Rrna Gene Pyrosequencing

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

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Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory

mSphere ◽

10.1128/msphere.00601-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Katja Engel ◽

Sian E. Ford ◽

Sara Coyotzi ◽

Jennifer McKelvie ◽

Nikitas Diomidis ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Corrosion Test ◽

Research Laboratory ◽

Bentonite Clay ◽

Rrna Gene ◽

Deep Geological Repository ◽

Geological Repository ◽

Underground Research Laboratory

ABSTRACT To assess the microbiology and corrosion potential of engineered components of a deep geological repository for long-term storage of high-level nuclear waste, the Materials Corrosion Test is being conducted at the Underground Research Laboratory in Grimsel, Switzerland. Modules containing metal coupons surrounded by highly compacted MX-80 bentonite, at two dry densities (1.25 and 1.50 g/cm3), were emplaced within 9-m-deep boreholes, and the first modules were retrieved after 13 months of exposure. Bentonite and associated module materials were sampled, and microbial communities and their distributions were assessed using 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) analysis. Borehole fluid was dominated by amplicon sequence variants (ASVs) affiliated with Desulfosporosinus and Desulfovibrio, which are putatively involved in sulfate reduction. The relative abundance of these ASVs was lower for samples from inside the borehole module, and they were almost undetectable in samples of the inner bentonite layer. The dominant ASV in case and filter sample sequence data was affiliated with Pseudomonas stutzeri, yet its relative abundance decreased in the inner layer samples. Streptomyces sp. ASVs were relatively abundant in all bentonite core sample data both prior to emplacement and after 13 months of exposure, presumably as metabolically inactive spores or extracellular “relic” DNA. PLFA concentrations in outer and inner layer bentonite samples suggested cellular abundances of 1 × 106 to 3 × 106 cells/g, with similar PLFA distributions within all bentonite samples. Our results demonstrate consistent microbial communities inside the saturated borehole module, providing the first evidence for microbial stability under conditions that mimic a deep geological repository. IMPORTANCE The Materials Corrosion Test in Grimsel Underground Research Laboratory, Switzerland, enables an evaluation of microbiological implications of bentonite clay at densities relevant for a deep geological repository. Our research demonstrates that after 13 months of exposure within a granitic host rock, the microbial 16S rRNA gene signatures of saturated bentonite clay within the modules were consistent with the profiles in the original clay used to pack the modules. Such results provide evidence that densities chosen for this emplacement test are refractory to microbial activity, at least on the relatively short time frame leading to the first time point sampling event, which will help inform in situ engineered barrier system science. This study has important implications for the design of deep geological repository sites under consideration for the Canadian Shield.

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Complementing 16S rRNA Gene Amplicon Sequencing with Total Bacterial Load To Infer Absolute Species Concentrations in the Vaginal Microbiome

mSystems ◽

10.1128/msystems.00777-19 ◽

2020 ◽

Vol 5 (2) ◽

Author(s):

Florencia A. Tettamanti Boshier ◽

Sujatha Srinivasan ◽

Anthony Lopez ◽

Noah G. Hoffman ◽

Sean Proll ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Bacterial Species ◽

Bacterial Load ◽

Amplicon Sequencing ◽

Individual Species ◽

Rrna Gene ◽

Vaginal Microbiome ◽

Bacterial Concentrations

ABSTRACT Whereas 16S rRNA gene amplicon sequencing quantifies relative abundances of bacterial taxa, variation in total bacterial load between samples restricts its ability to reflect absolute concentrations of individual bacterial species. Quantitative PCR (qPCR) can quantify individual species, but it is not practical to develop a suite of qPCR assays for every bacterium present in a diverse sample. We sought to determine the accuracy of an inferred measure of bacterial concentration using total bacterial load and relative abundance. We analyzed 1,320 samples from 20 women with a history of frequent bacterial vaginosis who self-collected vaginal swabs daily over 60 days. We inferred bacterial concentrations by taking the product of species relative abundance (assessed by 16S rRNA gene amplicon sequencing) and bacterial load (measured by broad-range 16S rRNA gene qPCR). Log10-converted inferred concentrations correlated with targeted qPCR (r = 0. 935, P < 2.2e–16) for seven key bacterial species. The mean inferred concentration error varied across bacteria, with rarer bacteria associated with larger errors. A total of 92% of the >0.5-log10 errors occurred when the relative abundance was <10%. Many errors occurred during early bacterial expansion from or late contraction to low abundance. When the relative abundance of a species is >10%, inferred concentrations are reliable proxies for targeted qPCR in the vaginal microbiome. However, targeted qPCR is required to capture bacteria at low relative abundance and is preferable for characterizing growth and decay kinetics of single species. IMPORTANCE Microbiome studies primarily use 16S rRNA gene amplicon sequencing to assess the relative abundance of bacterial taxa in a community. However, these measurements do not accurately reflect absolute taxon concentrations. We sought to determine whether the product of species’ relative abundance and total bacterial load measured by broad-range qPCR is an accurate proxy for individual species’ concentrations, as measured by taxon-specific qPCR assays. Overall, the inferred bacterial concentrations were a reasonable proxy of species-specific qPCR values, particularly when bacteria are present at a higher relative abundance. This approach offers an opportunity to assess the concentrations of bacterial species and how they change in a community over time without developing individual qPCR assays for each taxon.

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Corn oil supplementation enhances hydrogen use for biohydrogenation, inhibits methanogenesis, and alters fermentation pathways and the microbial community in the rumen of goats

Journal of Animal Science ◽

10.1093/jas/skz352 ◽

2019 ◽

Vol 97 (12) ◽

pp. 4999-5008 ◽

Cited By ~ 1

Author(s):

Xiu Min Zhang ◽

Rodolfo F Medrano ◽

Min Wang ◽

Karen A Beauchemin ◽

Zhi Yuan Ma ◽

...

Keyword(s):

Fatty Acids ◽

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Unsaturated Fatty Acids ◽

Corn Oil ◽

Nutrient Digestibility ◽

Rrna Gene ◽

Ch4 Emissions ◽

Gene Copies

Abstract Enteric methane (CH4) emissions are not only an important source of greenhouse gases but also a loss of dietary energy in livestock. Corn oil (CO) is rich in unsaturated fatty acid with >50% PUFA, which may enhance ruminal biohydrogenation of unsaturated fatty acids, leading to changes in ruminal H2 metabolism and methanogenesis. The objective of this study was to investigate the effect of CO supplementation of a diet on CH4 emissions, nutrient digestibility, ruminal dissolved gases, fermentation, and microbiota in goats. Six female goats were used in a crossover design with two dietary treatments, which included control and CO supplementation (30 g/kg DM basis). CO supplementation did not alter total-tract organic matter digestibility or populations of predominant ruminal fibrolytic microorganisms (protozoa, fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes), but reduced enteric CH4 emissions (g/kg DMI, −15.1%, P = 0.003). CO supplementation decreased ruminal dissolved hydrogen (dH2, P < 0.001) and dissolved CH4 (P < 0.001) concentrations, proportions of total unsaturated fatty acids (P < 0.001) and propionate (P = 0.015), and increased proportions of total SFAs (P < 0.001) and acetate (P < 0.001), and acetate to propionate ratio (P = 0.038) in rumen fluid. CO supplementation decreased relative abundance of family Bacteroidales_BS11_gut_group (P = 0.032), increased relative abundance of family Rikenellaceae (P = 0.021) and Lachnospiraceae (P = 0.025), and tended to increase relative abundance of genus Butyrivibrio_2 (P = 0.06). Relative abundance (P = 0.09) and 16S rRNA gene copies (P = 0.043) of order Methanomicrobiales, and relative abundance of genus Methanomicrobium (P = 0.09) also decreased with CO supplementation, but relative abundance (P = 0.012) and 16S rRNA gene copies (P = 0.08) of genus Methanobrevibacter increased. In summary, CO supplementation increased rumen biohydrogenatation by facilitating growth of biohydrogenating bacteria of family Lachnospiraceae and genus Butyrivibrio_2 and may have enhanced reductive acetogenesis by facilitating growth of family Lachnospiraceae. In conclusion, dietary supplementation of CO led to a shift of fermentation pathways that enhanced acetate production and decreased rumen dH2 concentration and CH4 emissions.

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Comparison of Euryarchaea Strains in the Guts and Food-Soil of the Soil-Feeding Termite Cubitermes fungifaber across Different Soil Types

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.3884-3892.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 3884-3892 ◽

Cited By ~ 50

Author(s):

S. E. Donovan ◽

K. J. Purdy ◽

M. D. Kane ◽

P. Eggleton

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rflp Analysis ◽

Methanogenic Archaea ◽

16S Rrna Gene Sequencing ◽

Nutrient Content ◽

Soil Types ◽

Rrna Gene ◽

Soil Communities ◽

Rrna Gene Sequencing

ABSTRACT Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

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Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols

Applied and Environmental Microbiology ◽

10.1128/aem.00726-09 ◽

2009 ◽

Vol 75 (17) ◽

pp. 5445-5450 ◽

Cited By ~ 64

Author(s):

Benjamin Nehmï¿½ ◽

Yan Gilbert ◽

Valï¿½rie Lï¿½tourneau ◽

Robert J. Forster ◽

Marc Veillette ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Denaturing Gradient Gel Electrophoresis ◽

Molecular Ecology ◽

Pcr Amplification ◽

Methanogenic Archaea ◽

Archaeal Community ◽

Pig Manure ◽

Rrna Gene ◽

Swine Confinement

ABSTRACT It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

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Determination of methanogenic Archaea abundance in a mesophilic biogas plant based on 16S rRNA gene sequence analysis

Canadian Journal of Microbiology ◽

10.1139/w10-021 ◽

2010 ◽

Vol 56 (5) ◽

pp. 440-444 ◽

Cited By ~ 20

Author(s):

I. Bergmann ◽

E. Nettmann ◽

K. Mundt ◽

M. Klocke

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Energy Production ◽

Methanogenic Archaea ◽

Biogas Plant ◽

Raw Material ◽

Gene Copy ◽

Rrna Gene ◽

Methane Formation ◽

Technical Equipment

Energy production of renewable raw material is of increasing importance for sustainable energy production. As an indispensable prerequirement for further upgrading of technical equipment and operation modes of biogas plants, a deeper knowledge of the microbial community responsible for methane formation is crucial. To overcome these limitations a mesophilic biogas plant converting pig manure, maize silage, and grains of crop was sampled and subsequently analysed by construction of a methanogenic Archaea specific 16S rRNA gene clone library combined with PCR–RFLP analysis and group-specific quantitative real-time PCR (qPCR). Seventy percent of all analysed clones belonged to the order Methanomicrobiales, whereas 13% belonged to Methanosarcinales, 6% belonged to the Methanobacteriales group, and 11% of all detected clones were assigned to the CA11 and Arch1 cluster. Comparable percentages were obtained with qPCR: 84% of all detected 16S rRNA gene copy numbers were affiliated with the Methanomicrobiales, while only 14% belonged to the Methanosarcinales and 2% to the Methanobacteriales order. In conclusion, both approaches detected similar archaeal groups and revealed nearly the same abundance, pointing to a predominance of hydrogenotrophic methanogens in the biogas plant.

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Pyrosequencing ofmcrAand Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

Applied and Environmental Microbiology ◽

10.1128/aem.02566-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 604-613 ◽

Cited By ~ 64

Author(s):

David Wilkins ◽

Xiao-Ying Lu ◽

Zhiyong Shen ◽

Jiapeng Chen ◽

Patrick K. H. Lee

Keyword(s):

Anaerobic Digestion ◽

16S Rrna ◽

16S Rrna Gene ◽

Methanogenic Archaea ◽

16S Rrna Gene Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Content Type ◽

The 16S Rrna Gene

ABSTRACTMethanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes.Methanobacteriales,Methanomicrobiales, andMethanosarcinaleswere detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrAgenes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales, while the other digesters were dominated by hydrogenotrophicMethanomicrobiales. The proposed euryarchaeotal orderMethanomassiliicoccalesand the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrAgenes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrAgene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrAtranscripts in digesters treating sludge and wastewater samples, supporting themcrAgene as a biomarker for methane yield.

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Handling of Spurious Sequences Affects the Outcome of High-Throughput 16S rRNA Gene Amplicon Profiling

10.21203/rs.3.rs-117421/v1 ◽

2020 ◽

Author(s):

Thomas Clavel ◽

Sandra Reitmeier ◽

Thomas CA Hitch ◽

Nicole Treichel ◽

Nikolaos Fikas ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Data Processing ◽

Relative Abundance ◽

Large Scale ◽

Peat Soil ◽

Rrna Gene ◽

Dnase Treatment

Abstract Background: 16S rRNA gene amplicon sequencing is a very popular approach for studying microbiomes. However, varying standards exist for sample and data processing and some basic concepts, such as the occurrence of spurious sequences, have not been investigated in a comprehensive manner. Methods: Using defined communities of bacteria in vitro and in vivo, we searched for sequences not matching the expected species (i.e., spurious taxa) and determined a minimum threshold of occurrence suitable for robust data analysis. The presence and origin of spurious taxa were investigated via large-scale amplicon queries and gut samples from germfree mice spiked with target mock DNA. We also assessed the effect of varying sequence-filtering stringency on diversity readouts in human fecal and peat soil communities. Our findings are based on data generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches.Results: 16S rRNA gene amplicon data-processing based on OTUs clustering and singleton removal, a commonly used approach that discards any taxa represented by only one sequence across all samples, delivered an average approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Most spurious taxa (approx. 70%) detected in simplified communities occurred in samples multiplexed in the same sequencing run and were present in only one of ten runs. DNase treatment of gut content from germfree mice partly helped to exclude spurious taxa from the analysis of spiked mock DNA, but was not necessary when applying the 0.25% relative abundance threshold. Using this cut-off improved the reproducibility of analysis, i.e., specifically by reducing variation in richness estimates by 38% compared with singleton filtering in a benchmarking experiment using six human fecal samples across seven sequencing runs. Beta-diversity analyses of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparing samples, highlighting the importance of carefully analyzing data before drawing conclusions. Conclusions: Handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. Applying a minimum relative abundance threshold between 0.10 and 0.30% is superior to the singleton removal approach, although study-specific analysis strategies may be needed depending on, for instance, the type of samples analyzed and the sequencing depth achieved. Additionally, we propose the concept of effective richness to facilitate the comparison of results across studies.

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