Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

Abstract Background 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1–V2 (V12) and the standard V3–V4 primer (V34) sets to optimize the gut microbiota analysis protocol. Results QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. Conclusions These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

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16S rRNA Gene Amplicon Sequencing of Gut Microbiota from Naked Carp (Gymnocypris przewalskii) in Qinghai Lake, China

Microbiology Resource Announcements ◽

10.1128/mra.00374-21 ◽

2021 ◽

Vol 10 (23) ◽

Author(s):

Fayan Wang ◽

Yu Liu ◽

Guangxin Li ◽

Xi Yang ◽

Qiang Gao

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Qinghai Lake ◽

Second Grade ◽

Rrna Gene ◽

Content Type ◽

Gymnocypris Przewalskii ◽

State Protection

Naked carp ( Gymnocypris przewalskii ) is a second-grade animal under state protection of China. We report 16S rRNA gene amplicon analysis of the gut microbiota of Gymnocypris przewalskii . The three most abundant phyla are Tenericutes , Proteobacteria , and Fusobacteria , and the six most abundant genera are Aeromonas , Clostridium , Cetobacterium , Shewanella , Prochlorococcus , and Vibrio .

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16S rRNA gene amplicon sequencing of gut microbiota in gestational diabetes mellitus and their correlation with disease risk factors

Journal of Endocrinological Investigation ◽

10.1007/s40618-021-01595-4 ◽

2021 ◽

Author(s):

J. Wei ◽

Y. Qing ◽

H. Zhou ◽

J. Liu ◽

C. Qi ◽

...

Keyword(s):

Diabetes Mellitus ◽

16S Rrna ◽

Gestational Diabetes ◽

Glucose Tolerance ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Gestational Diabetes Mellitus ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Strong Positive Correlation

Abstract Purpose Although the gut microbiota (GM) are associated with various diseases, their role in gestational diabetes mellitus (GDM) remains uncharacterized. Further study is urgently needed to expose the real relationship between GM and GDM. Methods We performed a prospective study in 33 pregnant Chinese individuals [15, GDM; 18, normal glucose tolerance (NGT)] to observe the fecal microbiota by 16S rRNA gene amplicon sequencing at 24–28 weeks of gestational age after a standard 75 g oral glucose tolerance test. Linear regression analysis was employed to assess the relationships between the GM and GDM clinical parameters. Results Sequencing showed no difference in the microbiota alpha diversity but a significant difference in the beta diversity between the GDM and NGT groups, with the relative abundances of Ruminococcus bromii, Clostridium colinum, and Streptococcus infantis being higher in the GDM group (P < 0.05). The quantitative PCR results validated the putative bacterial markers of R. bromii and S. infantis. Moreover, a strong positive correlation was found between S. infantis and blood glucose levels after adjusting for body mass index (P < 0.05). Conclusion Three abnormally expressed intestinal bacteria (R. bromii, C. colinum, and S. infantis) were identified in GDM patients. S. infantis may confer an increased risk of GDM. Hence, the GM may serve as a potential therapeutic target for GDM.

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16S rRNA Gene Amplicon Sequencing of Gut Microbiota in Three Species of Deep-Sea Fish in Suruga Bay, Japan

Microbiology Resource Announcements ◽

10.1128/mra.01260-20 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Toshihide Iwatsuki ◽

Takahiro Kanazawa ◽

Takato Ogasawara ◽

Kento Hosotani ◽

Karen Tsuchiya ◽

...

Keyword(s):

Sequence Analysis ◽

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Deep Sea ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Suruga Bay ◽

Sea Fish

ABSTRACT We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum Proteobacteria (genus Photobacterium), while one was dominated by the phyla Spirochaetes (genus Brevinema) and Tenericutes (unclassified Mycoplasmataceae).

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

Microbiome ◽

10.1186/s40168-021-01012-1 ◽

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.

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Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota

Scientific Reports ◽

10.1038/s41598-021-82726-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Francesco Durazzi ◽

Claudia Sala ◽

Gastone Castellani ◽

Gerardo Manfreda ◽

Daniel Remondini ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Shotgun Sequencing ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Experimental Conditions ◽

Rrna Gene Sequencing

AbstractIn this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

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

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

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.

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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

BMC Microbiology ◽

10.1186/s12866-021-02275-2 ◽

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.

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The protective effects of yellow tea extract against loperamide-induced constipation in mice

Food & Function ◽

10.1039/d0fo02969f ◽

2021 ◽

Author(s):

Pei-Qin Cao ◽

Xiu-Ping Li ◽

Jian Ou-Yang ◽

Rong-Gang Jiang ◽

Fang-Fang Huang ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Protective Effects ◽

Rrna Gene Sequencing ◽

Tea Extract

We evaluated the effects of yellow tea extract on relieving constipation induced by loperamide and evaluated the changes of gut microbiota based on 16S rRNA gene sequencing.

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

10.21203/rs.3.rs-321778/v1 ◽

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.

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