16S rRNA Gene Amplicon Sequencing Reveals Significant Changes in Microbial Compositions during Cyanobacteria-Laden Drinking Water Sludge Storage

2017 ◽  
Vol 51 (21) ◽  
pp. 12774-12783 ◽  
Author(s):  
Haiyan Pei ◽  
Hangzhou Xu ◽  
Jingjing Wang ◽  
Yan Jin ◽  
Hongdi Xiao ◽  
...  
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene

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 Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered 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. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

scholarly journals Detection of Legionella species, the influence of precipitation on the amount of Legionella DNA, and bacterial microbiome in aerosols from outdoor sites near asphalt roads in Toyama Prefecture, Japan

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jun-ichi Kanatani ◽  
Masanori Watahiki ◽  
Keiko Kimata ◽  
Tomoko Kato ◽  
Kaoru Uchida ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Indoor Environment ◽  
Geometric Mean ◽  
Amplicon Sequencing ◽  
Positive Sample ◽  
Outdoor Environment ◽  
Rrna Gene ◽  
Total Precipitation ◽  
Monthly Total Precipitation

Abstract Background Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites, such as buildings and private homes, using amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing. Results Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = − 0.29, P < 0.01) before the sampling day, respectively. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features. Conclusions DNA from Legionella species was widely present in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.

scholarly journals Nocardioides hungaricus sp. nov., isolated from a drinking water supply system

2011 ◽  
Vol 61 (3) ◽  
pp. 549-553 ◽  
Author(s):  
Erika M. Tóth ◽  
Zsuzsa Kéki ◽  
Judit Makk ◽  
Zalán G. Homonnay ◽  
Károly Márialigeti ◽  
...  
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Supply ◽  
Gene Sequence ◽  
Supply System ◽  
Drinking Water Supply ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Drinking Water Supply System

Three Gram-positive, rod-shaped bacterial strains were isolated from the drinking water supply system of the Hungarian capital, Budapest. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparison revealed that the isolates represented a distinct cluster within the clade of the genus Nocardioides and were most closely related to Nocardioides pyridinolyticus OS4T, Nocardioides aquiterrae GW-9T, Nocardioides sediminis MSL-01T and N. hankookensis DS-30T. The peptidoglycan based on ll-2,6-diaminopimelic acid, the major menaquinone MK-8(H4), the cellular fatty acid profile with iso-C16 : 0 and anteiso-C17 : 0 as predominating components and the DNA G+C content of 71.4 mol% (strain 1RaM5-12T) were consistent with the affiliation of the isolates to the genus Nocardioides. Because of differences in physiological characteristics, matrix-assisted laser-desorption/ionization time-of-flight mass spectra of protein extracts, PvuII RiboPrinter patterns and 96.1 % 16S rRNA gene sequence similarity between strain 1RaM5-12T and its closest phylogenetic neighbour, N. pyridinolyticus OS4T, a novel species, Nocardioides hungaricus sp. nov., is proposed. The type strain is 1RaM5-12T (=DSM 21673T =NCAIM 02330T).

scholarly journals Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant

2012 ◽  
Vol 62 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Ivone Vaz-Moreira ◽  
Vânia Figueira ◽  
Ana R. Lopes ◽  
Alexandre Lobo-da-Cunha ◽  
Cathrin Spröer ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Treatment ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Treatment Plant ◽  
Drinking Water Treatment ◽  
Water Treatment Plant ◽  
Rrna Gene ◽  
Rrna Gene Sequence

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22T, was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15–37 °C, at pH 7–10 and with <8 % (w/v) NaCl (optimum growth: 30 °C, pH 7–8 and 1–3 % NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22T was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162T (98.5 % 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2T (97.9 %), Bacillus infantis SMC 4352-1T (97.4 %), Bacillus firmus IAM 12464T (96.8 %) and Bacillus muralis LMG 20238T (96.8 %). DNA–DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22T from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22T ( = DSM 23494T = NRRL B-59432T = LMG 25783T).

scholarly journals Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

2021 ◽  
Author(s):  
Seppo Virtanen ◽  
Schahzad Saqib ◽  
Tinja Kanerva ◽  
Pekka Nieminen ◽  
Ilkka Kalliala ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Extensive Literature ◽  
Metagenomic Sequencing ◽  
Text Extraction ◽  
Healthy Women ◽  
Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

scholarly journals High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

2019 ◽  
Vol 47 (18) ◽  
pp. e103-e103 ◽  
Author(s):  
Benjamin J Callahan ◽  
Joan Wong ◽  
Cheryl Heiner ◽  
Steve Oh ◽  
Casey M Theriot ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Full Complement ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

scholarly journals Interpretations of Environmental Microbial Community Studies Are Biased by the Selected 16S rRNA (Gene) Amplicon Sequencing Pipeline

2020 ◽  
Vol 11 ◽  
Author(s):  
Daniel Straub ◽  
Nia Blackwell ◽  
Adrian Langarica-Fuentes ◽  
Alexander Peltzer ◽  
Sven Nahnsen ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Community Studies

scholarly journals Prokaryotic Community Structures in a Thermophilic Anaerobic Digestion Reactor Converting Poly(l-Lactic Acid) for a Long Period Revealed by 16S rRNA Gene Amplicon Sequencing

2019 ◽  
Vol 8 (29) ◽  
Author(s):  
Takeshi Yamada ◽  
Masako Hamada ◽  
Misaki Kurobe ◽  
Jun Harada ◽  
Surya Giri ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Anaerobic Digestion ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Data ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Thermophilic Anaerobic Digestion ◽  
Taxonomic Class ◽  
Class Level

Little information on poly(l-lactic acid) (PLLA) treatment-associated microbiota in thermophilic anaerobic digestion reactors is available. Here, we provide 16S rRNA gene sequence data on microbiota in a thermophilic anaerobic digestion reactor converting PLLA to methane for 336 days. Data comprising 99,566 total high-quality reads were tabulated at the taxonomic class level.

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