Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the Illumina platform

As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner -- with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50\% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition. (version 1.1 resubmitted to PLOS one 2014-Sept-08)

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Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

Frontiers in Microbiology ◽

10.3389/fmicb.2021.740932 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hannah E. Epstein ◽

Alejandra Hernandez-Agreda ◽

Samuel Starko ◽

Julia K. Baum ◽

Rebecca Vega Thurber

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Beta Diversity ◽

Sequence Data ◽

Alpha Diversity ◽

Gene Profiling ◽

Rrna Gene ◽

Sequencing Platform ◽

Alpha And Beta Diversity ◽

Diversity Metrics

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.

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Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

PLoS ONE ◽

10.1371/journal.pone.0116955 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0116955 ◽

Cited By ~ 165

Author(s):

Lucas Sinclair ◽

Omneya Ahmed Osman ◽

Stefan Bertilsson ◽

Alexander Eiler

Keyword(s):

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Microbial Community Composition ◽

Rrna Gene ◽

Illumina Platform

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Evaluating the Impact of Wastewater Effluent on Microbial Communities in the Panke, an Urban River

Water ◽

10.3390/w11050888 ◽

2019 ◽

Vol 11 (5) ◽

pp. 888 ◽

Cited By ~ 2

Author(s):

Marcella Nega ◽

Burga Braun ◽

Sven Künzel ◽

Ulrich Szewzyk

Keyword(s):

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Sampling Site ◽

River Sediments ◽

Microbial Community Composition ◽

Rrna Gene ◽

Sequencing Data ◽

Alpha And Beta Diversity ◽

The Impact

Pharmaceuticals are consumed in high amounts and can enter as emerging organic compounds in surface waters as they are only partially retained in wastewater treatment plants (WWTPs). Receiving pharmaceuticals may burden the aquatic environment, as they are designed to be bioactive even at low concentrations. Sediment biofilm populations were analyzed in river sediments due to the exposure of an inflow of WWTP effluents. Illumina MiSeq 16S rRNA gene amplicon sequencing was performed of 108 sediment samples, which were taken from multiple cores within three sampling locations in the Panke River, with one sampling site located downstream of the inflow. Sequencing data were processed to infer microbial community structure in samples concerning the environmental variables, such as micropollutants and physicochemical parameters measured for each core. More than 25 different micropollutants were measured in pore water samples, in which bezafibrate, clofibric acid, carbamazepine, and diclofenac were detected at high concentrations. Bacterial 16S rRNA gene amplicons revealed Nitrospirae, Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Bacteroidetes, and Ignavibacteriae as the most abundant groups in the samples. Differences in microbial community composition were observed with respect to micropollutants. However, our findings revealed that the composition of the microbial community was not only governed by the effluent. The significant changes in the alpha- and beta-diversity were explained by phenobarbital and SO42−, which did not originate from the WWTP indicating that more unobserved factors are also likely to play a role in affecting the biofilm community’s composition.

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AB1232 ORAL DYSBIOSIS REFLECTS THE IMMUNOLOGICAL ALTERATION OF RA REGARDING TO ACPA AND HLA DRB1*SE: NAGASAKI ISLAND STUDY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5147 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1907.2-1907

Author(s):

Y. Tsuji ◽

M. Tamai ◽

S. Morimoto ◽

D. Sasaki ◽

M. Nagayoshi ◽

...

Keyword(s):

Beta Diversity ◽

Healthy Subjects ◽

Microbial Community Composition ◽

Alpha Diversity ◽

Enzyme Linked Immunosorbent Assay ◽

Rrna Gene ◽

Oral Microbiota ◽

Unifrac Distance ◽

Alpha And Beta Diversity ◽

The Difference

Background:Anti-citrullinated protein antibody (ACPA) production is observed in several organs even prior to the onset of rheumatoid arthritis (RA), and oral mucosa is considered to be one of the important tissues. The presence of HLA-DRB1*SE closely associates with ACPA production. Saliva is considered to reflect the oral microbiota including periodontal disease. Alteration of oral microbiota of RA becomes to be normalized by DMARDs treatment, however, the interaction of HLA-DRB1*SE, ACPA and oral microbiota of RA patients remains to be elucidated.Objectives:The Nagasaki Island Study, which had started in 2014 collaborating with Goto City, is intended for research of the preclinical stage of RA, including ACPA/HLA genotype screening and ultrasound and magnetic resonance imaging examinations in high-risk subjects. Using the samples accumulated in this cohort, we have tried to investigate the difference of oral microbiota among RA patients and healthy subjects regarding to ACPA and HLA-DRB1*SE.Methods:Blood and salivary samples were obtained from 1422 subjects out of 4276 who have participated in the Nagasaki Island Study from 2016 to 2018. ACPA positivity was 1.7 % in total. Some of RA patients resided in Goto City participated in the Nagasaki Island Study. At this point, we selected 291 subjects, who were ACPA positive non-RA healthy subjects (n=22) and patients with RA (n=33, 11 subjects were ACPA positive and 22 ACPA negative respectively) as the case, age and gender matched ACPA negative non-RA healthy subjects (n=236) as the control. ACPA was measured by an enzyme-linked immunosorbent assay, and HLA genotyping was quantified by next-generation sequencing (Ref.1). The operational taxonomic unit (OUT) analysis using 16S rRNA gene sequencing were performed. The richness of microbial diversity within-subject (alpha diversity) was scaled via Shannon entropy. The dissimilarity between microbial community composition was calculated using Bray-Curtis distance as a scale, and differences between groups (beta diversity) were tested by permutational multivariate analysis of variance (PERMANOVA). In addition, UniFrac distance calculated in consideration of the distance on the phylogenetic tree were performed.Results:Median age 70 y.o., % Female 58.8 %. Among RA and non-RA subjects, not alpha diversity but beta diversity was statistically significance (p=0.022, small in RA). In RA subjects, both alpha and beta diversity is small (p<0.0001), especially significant in ACPA positive RA (Figure 1). Amongt RA subjects, presence of HLA-DRB1*SE did not show the difference but the tendency of being small of alpha diversity (p=0.29).Conclusion:Our study has suggested for the first time the association of oral microbiota alteration with the presence of ACPA and HLA-DRB1*SE. Oral dysbiosis may reflect the immunological status of patients with RA.References:[1]Kawaguchi S, et al. Methods Mol Biol 2018;1802: 22Disclosure of Interests:None declared

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PSIX-3 Comparison of 16S rRNA gene profiles of rumen microbiome from Raramuri Criollo, European and Criollo x European lineages

Journal of Animal Science ◽

10.1093/jas/skab235.789 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 441-442

Author(s):

Adrian Maynez-Perez ◽

Francisco Jahuey-Martinez ◽

Jose A Martinez-Quintana ◽

Michael E Hume ◽

Robin C Anderson ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Beta Diversity ◽

Chihuahuan Desert ◽

Reference Database ◽

Rrna Gene ◽

Linear Discriminant ◽

Microbiome Composition ◽

Northern Mexico ◽

Rumen Microbiome

Abstract Raramuri Criollo cattle from the Chihuahuan desert in northern Mexico have been described as an ecological ecotype due to their enormous advantage in land grass utilization and their capacity to diversify their diet with cacti, forbs and woody plants. This diversification in diet utilization, could reflect upon their microbiome composition. The aim of this study was to characterize the rumen microbiome of Raramuri criollo cattle and to compare it to other lineages that graze in the same area. A total of 28 cows representing three linages [Criollo (n = 13), European (n = 9) and Criollo x European Crossbred (n = 6)] were grazed without supplementation for 45 days. DNA was extracted from ruminal samples and the V4 region of the 16S rRNA gene was sequenced on an Illumina platform. Data were analyzed with the QIIME2 software package and DADA2 plugin and the amplicon sequence variants were taxonomically classified with naïve Bayesian using the SILVA 16S rRNA gene reference database (version 132). Statistical analysis was performed by ANOVA and PERMANOVA for alpha and beta diversity indexes, respectively, and the non-strict version of linear discriminant analysis effect size (LEfSe) was used to determine significantly different taxa among lineages. Differences in beta diversity indexes (P < 0.05) were found in ruminal microbiome composition between Criollo and European groups, whereas the Crossbred showed intermediate values when compared to the pure breeds (Table 1). LEfSe analysis identified a total of 20 bacterial groups that explained differences between lineages, including one for Crossbreed, ten for European and nine for Criollo. These results show ruminal microbiome differences between Raramuri criollo cattle and the mainstream European breeds used in the northern Mexico Chihuahuan desert and reflect that those differences could be a consequence of dissimilar grazing behavior.

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Epipelic cyanobacterial diversity in Pinang River basin, Malaysia, revealed by 16S-based metagenomic approach

Algologia ◽

10.15407/alg31.01.093 ◽

2021 ◽

Vol 31 (1) ◽

pp. 93-113

Author(s):

A.R. Nur Fadzliana ◽

◽

W.O. Wan Maznah ◽

S.A.M. Nor ◽

Choon Pin Foong ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

River Basin ◽

Sequence Data ◽

Diversity Index ◽

Saline Water ◽

Illumina Miseq ◽

Rrna Gene ◽

Cyanobacterial Community ◽

Upstream Station

Cyanobacteria are the most widespread group of photosynthetic prokaryotes. They are primary producers in a wide variety of habitats and are able to thrive in harsh environments, including polluted waters; therefore, this study was conducted to explore the cyanobacterial populations inhabiting river tributaries with different levels of pollution. Sediment samples (epipelon) were collected from selected tributaries of the Pinang River basin. Air Terjun (T1) and Air Itam rivers (T2) represent the upper streams of Pinang River basin, while Dondang (T3) and Jelutong rivers (T4) are located at in the middle of the river basin. The Pinang River (T5) is located near the estuary and is subjected to saline water intrusion during high tides. Cyanobacterial community was determined by identifying the taxa via 16S rRNA gene amplicon sequence data. 16S rRNA gene amplicons generated from collected samples were sequenced using illumina Miseq, with the targeted V3 and V4 regions yielding approximately 1 mln reads per sample. Synechococcus, Phormidium, Arthronema and Leptolyngbya were found in all samples. Shannon-Weiner diversity index was highest (H’ = 1.867) at the clean upstream station (T1), while the moderately polluted stream (T3) recorded the lowest diversity (H’ = 0.399), and relatively polluted stations (T4 and T5) recorded fairly high values of H’. This study provides insights into the cyanobacterial community structure in Pinang River basin via cultivation-independent techniques using 16S rRNA gene amplicon sequence. Occurrence of some morphospecies at specific locations showed that the cyanobacterial communities are quite distinct and have specific ecological demands. Some species which were ubiquitous might be able to tolerate varied environmental conditions.

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Three novel lineages of ‘Candidatus Liberibacter africanus’ associated with native rutaceous hosts of Trioza erytreae in South Africa

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.069864-0 ◽

2015 ◽

Vol 65 (Pt_2) ◽

pp. 723-731 ◽

Cited By ~ 38

Author(s):

Ronel Roberts ◽

Emma T. Steenkamp ◽

Gerhard Pietersen

Keyword(s):

South Africa ◽

Phylogenetic Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Nucleotide Sequence Data ◽

Trioza Erytreae

Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’ (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp. clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.

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Fecal Short-Chain Fatty Acids Are Not Predictive of Colonic Tumor Status and Cannot Be Predicted Based on Bacterial Community Structure

mBio ◽

10.1128/mbio.01454-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 5

Author(s):

Marc A. Sze ◽

Begüm D. Topçuoğlu ◽

Nicholas A. Lesniak ◽

Mack T. Ruffin ◽

Patrick D. Schloss

Keyword(s):

Colorectal Cancer ◽

Fatty Acids ◽

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Short Chain Fatty Acids ◽

Tumor Burden ◽

Short Chain ◽

Rrna Gene ◽

Chain Fatty Acids

ABSTRACT Colonic bacterial populations are thought to have a role in the development of colorectal cancer with some protecting against inflammation and others exacerbating inflammation. Short-chain fatty acids (SCFAs) have been shown to have anti-inflammatory properties and are produced in large quantities by colonic bacteria that produce SCFAs by fermenting fiber. We assessed whether there was an association between fecal SCFA concentrations and the presence of colonic adenomas or carcinomas in a cohort of individuals using 16S rRNA gene and metagenomic shotgun sequence data. We measured the fecal concentrations of acetate, propionate, and butyrate within the cohort and found that there were no significant associations between SCFA concentration and tumor status. When we incorporated these concentrations into random forest classification models trained to differentiate between people with healthy colons and those with adenomas or carcinomas, we found that they did not significantly improve the ability of 16S rRNA gene or metagenomic gene sequence-based models to classify individuals. Finally, we generated random forest regression models trained to predict the concentration of each SCFA based on 16S rRNA gene or metagenomic gene sequence data from the same samples. These models performed poorly and were able to explain at most 14% of the observed variation in the SCFA concentrations. These results support the broader epidemiological data that questions the value of fiber consumption for reducing the risks of colorectal cancer. Although other bacterial metabolites may serve as biomarkers to detect adenomas or carcinomas, fecal SCFA concentrations have limited predictive power. IMPORTANCE Considering that colorectal cancer is the third leading cancer-related cause of death within the United States, it is important to detect colorectal tumors early and to prevent the formation of tumors. Short-chain fatty acids (SCFAs) are often used as a surrogate for measuring gut health and for being anticarcinogenic because of their anti-inflammatory properties. We evaluated the fecal SCFA concentrations of a cohort of individuals with different colonic tumor burdens who were previously analyzed to identify microbiome-based biomarkers of tumors. We were unable to find an association between SCFA concentration and tumor burden or use SCFAs to improve our microbiome-based models of classifying people based on their tumor status. Furthermore, we were unable to find an association between the fecal community structure and SCFA concentrations. Our results indicate that the association between fecal SCFAs, the gut microbiome, and tumor burden is weak.

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Prokaryotic taxonomy in the sequencing era and the role of MLSA in classification

Microbiology Australia ◽

10.1071/ma11066 ◽

2011 ◽

Vol 32 (2) ◽

pp. 66 ◽

Cited By ~ 4

Author(s):

Peter Kampfer ◽

Stefanie P Glaeser

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

16S Rrna Gene Sequencing ◽

Initial Step ◽

Rrna Gene ◽

Sequence Analyses ◽

Phenotypic Characterisation ◽

Novel Taxa ◽

Rrna Gene Sequencing

The initial step in prokaryote species and genera descriptions is now largely based on the 16S rRNA gene sequencing approach followed often by a very restricted additional phenotypic characterisation of the representatives of the potential novel taxa. Despite the advantages of the sequence-based approaches, there appears to be a tendency to classify new species on the basis of comparative sequence analyses of 16S rRNA gene sequences and other gene sequence data (multilocus sequence analyses, MLSA), contrary to the indications of other data. However, the biological meaning behind these sequence data is not always clear, and one should be careful with comprehensive taxonomic rearrangements until there is better insight of these data.

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16S rRNA Gene Amplicon Profiling of Baby and Adult Captive Elephants in Thailand

Microbiology Resource Announcements ◽

10.1128/mra.00248-20 ◽

2020 ◽

Vol 9 (24) ◽

Author(s):

Sangam Kandel ◽

Supaphen Sripiboon ◽

Piroon Jenjaroenpun ◽

David W. Ussery ◽

Intawat Nookaew ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Diversity ◽

Sequence Data ◽

Rrna Gene ◽

Data Set ◽

Geographical Locations

ABSTRACT Here, we present a 16S rRNA gene amplicon sequence data set and profiles demonstrating the bacterial diversity of baby and adult elephants from four different geographical locations in Thailand. The dominant phyla among baby and adult elephants were Bacteroidetes, Firmicutes, Proteobacteria, Kiritimatiellaeota, Euryarchaeota, and Tenericutes.

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