scholarly journals Interpretations of microbial community studies are biased by the selected 16S rRNA gene amplicon sequencing pipeline

2019 ◽  
Author(s):  
Daniel Straub ◽  
Nia Blackwell ◽  
Adrian Langarica Fuentes ◽  
Alexander Peltzer ◽  
Sven Nahnsen ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Environmental Samples ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Rrna Gene Sequencing ◽  
Diversity Estimates

AbstractOne of the major methods to identify microbial community composition, to unravel microbial population dynamics, and to explore microbial diversity in environmental samples is DNA- or RNA-based 16S rRNA (gene) amplicon sequencing. Subsequent bioinformatics analyses are required to extract valuable information from the high-throughput sequencing approach. However, manifold bioinformatics tools complicate their choice and might cause differences in data interpretation, making the selection of the pipeline a crucial step.Here, we compared the performance of most widely used 16S rRNA gene amplicon sequencing analysis tools (i.e. Mothur, QIIME1, QIIME2, and MEGAN) using mock datasets and environmental samples from contrasting terrestrial and freshwater sites. Our results showed that QIIME2 outcompeted all other investigated tools in sequence recovery (>10 times less false positives), taxonomic assignments (>22% better F-score) and diversity estimates (>5% better assessment), while there was still room for improvement e.g. imperfect sequence recovery (recall up to 87%) or detection of additional false sequences (precision up to 72%). Furthermore, we found that microbial diversity estimates and highest abundant taxa varied among analysis pipelines (i.e. only one in five genera was shared among all analysis tools) when analyzing environmental samples, which might skew biological conclusions.Our findings were subsequently implemented in a high-performance computing conformant workflow following the FAIR (Findable, Accessible, Interoperable, and Re-usable) principle, allowing reproducible 16S rRNA gene amplicon sequence analysis starting from raw sequence files. Our presented workflow can be utilized for future studies, thereby facilitating the analysis of high-throughput DNA- or RNA-based 16S rRNA (gene) sequencing data substantially.ImportanceMicroorganisms play an essential role in biogeochemical cycling events across the globe. Phylogenetic marker gene analysis is a widely used method to explore microbial community dynamics in space and time, to predict the ecological relevance of microbial populations, or to identify microbial key players in biogeochemical cycles. Several computational analysis methods were developed to aid 16S rRNA gene analysis but choosing the best method is not trivial. In this study, we compared popular analysis methods (i.e. Mothur, QIIME1 and 2, and MEGAN) using samples with known microbial composition (i.e. mock community samples) and environmental samples from contrasting habitats (i.e. groundwater, soil, sediment, and river water). Our findings provide guidance for choosing the currently optimal 16S rRNA gene sequencing analysis method and we implemented our recommended pipeline into a reproducible workflow, which follows highest bioinformatics standards and is open source and free to use.

scholarly journals Survey of Full Scale Side-Stream EBPR Facilities and Comparison with Conventional EBPR: Process Stability, Kinetics and Microbial Ecology

2018 ◽  
Author(s):  
Annalisa Onnis-Hayden ◽  
Varun Srinivasan ◽  
Nicholas B. Tooker ◽  
Guangyu Li ◽  
Dongqi Wang ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Systematic Evaluation ◽  
Rrna Gene ◽  
Process Stability ◽  
Side Stream ◽  
Rrna Gene Sequencing

Side-stream EBPR process (S2EBPR) is a new alternative to address the common challenges in EBPR related to weak wastewater influent and to improve EBPR process stability. A systematic evaluation and comparison of the process performance and microbial community structure between four S2EBPR with conventional EBPR configurations in US was conducted. The statistical analysis suggested higher performance stability in S2EBPR than the conventional EBPRs, although possible bias is recognized due to variations in the target permit levels and plant-specific factors among the plants. Total and known PAOs and GAOs abundance and identities were investigated with FISH, DAPI, 16S rRNA gene sequencing and Raman microspectroscopy. The results suggested comparable relative PAO and Candidatus Accumulibacter abundances in S2EBPR and conventional EBPR systems. Tetrasphaera, a putative PAO, was also found at similar abundance in S2EBPR as in conventional facilities, whereas the relative abundance of known GAOs was lower in S2EBPR than those typically seen at conventional EBPRs. Microbial community analyses via 16S rRNA gene amplicon sequencing revealed differences in the community phylogenetic fingerprints between S2EBPR and conventional plants. Shannon and Inverse Simpson indices, which are combined measures of richness and evenness evaluation of the microbial communities, suggested that the microbial diversity in S2EBPR plants were higher than those in conventional EBPRs.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals First case of an invasive Bacteroides dorei infection detected in a patient with a mycotic aortic aneurysm—raising a rebellion of major indigenous bacteria in humans: a case report and review

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Takayuki Matsuoka ◽  
Takuya Shimizu ◽  
Tadanori Minagawa ◽  
Wakiko Hiranuma ◽  
Miki Takeda ◽  
...  
Keyword(s):  
Aortic Aneurysm ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Resected Specimen ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
First Case ◽  
Rrna Gene Sequencing

Abstract Background Bacteroides dorei is an anaerobic gram-negative bacterium first described in 2006. Because of the high similarity in mass spectra between B. dorei and Bacteroides vulgatus, discriminating between these species is arduous in clinical practice. In recent decades, 16S rRNA gene sequencing has been a complementary method for distinguishing taxonomically close bacteria, including B. dorei and B. vulgatus, at the genus and species levels. Consequently, B. dorei has been shown to contribute to some diseases, including type 1 autoimmune diabetes mellitus and atherosclerotic diseases. However, there are no reports on invasive infectious diseases caused by B. dorei. This report describes the first case of direct invasion and colonisation of human tissue by B. dorei, thus providing a warning regarding the previously proposed application of B. dorei as a live biotherapeutic for atherosclerotic diseases. Case presentation A 78-year-old Japanese man complained of intermittent chest/back pain and was diagnosed with a mycotic thoracic aortic aneurysm by enhanced computed tomography on admission. Despite strict blood pressure control and empirical antibiotic therapy, the patient’s condition worsened. To prevent aneurysmal rupture and eliminate infectious foci, the patient underwent surgical treatment. The resected specimen was subjected to tissue culture and 16S rRNA gene sequencing analysis to identify pathogenic bacteria. A few days after the surgery, culture and sequencing results revealed that the pathogen was B. dorei/B. vulgatus and B. dorei, respectively. The patient was successfully treated with appropriate antibacterial therapy and after improvement, was transferred to another hospital for rehabilitation on postoperative day 34. There was no recurrence of infection or aneurysm after the patient transfer. Conclusions This report describes the first case of invasive infectious disease caused by B. dorei, casting a shadow over its utilisation as a probiotic for atherosclerotic diseases.

scholarly journals Arsenic mobilization in a high arsenic groundwater revealed by metagenomic and Geochip analyses

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhou Jiang ◽  
Ping Li ◽  
Yanhong Wang ◽  
Han Liu ◽  
Dazhun Wei ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Arsenic Mobilization ◽  
Rrna Gene Sequencing ◽  
High Arsenic ◽  
High Arsenic Groundwater

Abstract Microbial metabolisms of arsenic, iron, sulfur, nitrogen and organic matter play important roles in arsenic mobilization in aquifer. In this study, microbial community composition and functional potentials in a high arsenic groundwater were investigated using integrated techniques of RNA- and DNA-based 16S rRNA gene sequencing, metagenomic sequencing and functional gene arrays. 16S rRNA gene sequencing showed the sample was dominated by members of Proteobacteria (62.3–75.2%), such as genera of Simplicispira (5.7–6.7%), Pseudomonas (3.3–5.7%), Ferribacterium (1.6–4.4%), Solimonas (1.8–3.2%), Geobacter (0.8–2.2%) and Sediminibacterium (0.6–2.4%). Functional potential analyses indicated that organics degradation, assimilatory sulfate reduction, As-resistant pathway, iron reduction, ammonification, nitrogen fixation, denitrification and dissimilatory nitrate reduction to ammonia were prevalent. The composition and function of microbial community and reconstructed genome bins suggest that high level of arsenite in the groundwater may be attributed to arsenate release from iron oxides reductive dissolution by the iron-reducing bacteria, and subsequent arsenate reduction by ammonia-producing bacteria featuring ars operon. This study highlights the relationship between biogeochemical cycling of arsenic and nitrogen in groundwater, which potentially occur in other aquifers with high levels of ammonia and arsenic.

scholarly journals The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Wei Song ◽  
Lingzhi Li ◽  
Hongliang Huang ◽  
Keji Jiang ◽  
Fengying Zhang ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Communities ◽  
Antarctic Fish ◽  
16S Rrna Gene Sequencing ◽  
Physiological Characteristics ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Gut Microbial Community

Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics.

scholarly journals Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

2010 ◽  
Vol 4 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Jens JØrgen Christensen ◽  
Brita Bruun ◽  
Ute Wolff Sönksen ◽  
Lisbeth Nielsen ◽  
Annemarie Hesselbjerg ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Vitek 2 ◽  
Rrna Gene Sequencing

scholarly journals Bacterial composition of nasal discharge in children based on highly accurate 16S rRNA gene sequencing analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaoru Haro ◽  
Midori Ogawa ◽  
Mitsumasa Saito ◽  
Koichi Kusuhara ◽  
Kazumasa Fukuda
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Nasal Discharge ◽  
Rrna Gene ◽  
Respiratory Pathogens ◽  
Sequencing Analysis ◽  
Bacterial Cells ◽  
Rrna Gene Sequencing

AbstractNasopharyngeal colonization by bacteria is a prerequisite for progression to respiratory disease and an important source of horizontal spread within communities. We aimed to perform quantitative analysis of the bacterial cells and reveal the microbiota of the nasal discharge in children at the species level based on highly accurate 16S rRNA gene sequencing. This study enrolled 40 pediatric patients with rhinorrhea. The bacterial cells in the nasal discharge were counted by epifluorescence microscopic analysis. The microbiota was analyzed by using the 16S rRNA gene clone library sequencing method. We demonstrated that a high abundance (median 2.2 × 107 cells/mL) of bacteria was contained in the nasal discharge of children. Of the 40 samples, 37 (92.5%) were dominated by OTUs corresponding to Haemophilus aegyptius/influenzae, Moraxella catarrhalis/nonliquefaciens, or Streptococcus pneumoniae. These samples showed higher cell abundance and lower alpha diversity than the remaining three samples in which the other bacteria coexisted. In addition, 12 sequences with low homology to type strains were considered as previously unknown bacterial lineages. In conclusion, the nasal discharge of most young children contains a large amount of respiratory pathogens and several unknown bacteria, which could not only cause endogenous infection but also be a source of transmission to others.

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