Comparative analysis of microbiome of bladder cancer patients

2020 ◽  
Author(s):  
Ayesha Ishaq ◽  
Saman Zahid ◽  
Salma Mukhtar ◽  
Muhammad Kamran Azim ◽  
Saleem Akhtar ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
16S Rrna ◽  
Bacterial Diversity ◽  
Cancer Patients ◽  
Beta Diversity ◽  
Control Sample ◽  
Diagnostic Tools ◽  
Rrna Gene ◽  
Male And Female ◽  
Alpha And Beta Diversity

Abstract Background: It has been studied that the urinary tract, which was once considered to be sterile retains a unique microbiome. The current study was performed to explore the microbiome of male and female cancerous bladder tissue, including 01 control sample and 09 cancer samples using 16S rRNA gene sequencing. In previous studies, the significance of microbiome has been found to be associated with the bladder cancer. For diversity analysis, V3-V4 regions of 16S rRNA were used for PCR and later sequencing was carried out through Illumina, Miseq platform. The metadata generated was analyzed on QIIME 1.9.1.Result: The bacterial diversity detected showed that five of the phyla; namely Proteobacteria (38.1%), Firmicutes (37.8%), Actinobacteria (5.9%), Thermi (4.9%) and Tenericutes (2.5%) were more abundant in all samples as compared to other phyla. The genera found in all samples were Enterobacter (18.3%), Bacillus (13.9%), Meiothermus (4.9%), Methylotenera (1.2%), Ralstonia (3.6%) and Streptocococcus (1.4%). Ralstonia and Streptococcus were absent in BLC 10 and BLC 2, respectively, while present in the rest. The results of alpha and beta diversity showed that female samples had more bacterial diversity and uniformity as compared to male samples. Conclusion: The present study used biopsy samples of newly diagnosed cancer patients without taking into account any treatment given to the cancer patients. Our analysis showed insignificant differences in alpha and beta diversity of male and female samples. The genus Meiothermus detected in this study was firstly reported in a bladder microbiome analysis. The data generated from such preliminary futuristic study can help in devising new diagnostic tools and therapies.

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

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.

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

2014 ◽  
Author(s):  
Lucas Sinclair ◽  
Omneya Ahmed Osman ◽  
Stefan Bertilsson ◽  
Alexander Eiler
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Beta Diversity ◽  
Sequence Data ◽  
Soda Lakes ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Clustering Methods ◽  
Illumina Platform ◽  
Alpha And Beta Diversity

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)

scholarly journals Choice of 16S ribosomal RNA primers affects the microbiome analysis in chicken ceca

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadia Darwish ◽  
Jonathan Shao ◽  
Lori L. Schreier ◽  
Monika Proszkowiec-Weglarz
Keyword(s):  
16S Rrna ◽  
Dna Sequences ◽  
Bacterial Communities ◽  
Beta Diversity ◽  
Taxonomic Composition ◽  
Rrna Gene ◽  
Unifrac Distance ◽  
Variable Regions ◽  
Alpha And Beta Diversity ◽  
Predicted Function

AbstractWe evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4–V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1–V3(1) and (2), and V3–V4 and V3–V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.

scholarly journals The Effect of Common Viral Inactivation Techniques on 16S rRNA Amplicon-Based Analysis of the Gut Microbiota

2021 ◽  
Vol 9 (8) ◽  
pp. 1755
Author(s):  
Zachary McAdams ◽  
Kevin Gustafson ◽  
Aaron Ericsson
Keyword(s):  
16S Rrna ◽  
Gut Microbiome ◽  
Beta Diversity ◽  
Sodium Dodecyl ◽  
Rrna Gene ◽  
Fecal Dna ◽  
Viral Inactivation ◽  
Fecal Samples ◽  
Alpha And Beta Diversity ◽  
Microbiome Analysis

Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched fecal samples from sixteen female CD-1 mice of the same GM background prior to fecal DNA extraction. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA. Treatment-dependent effects on DNA yield, genus-level taxonomic abundance, and alpha and beta diversity metrics were assessed. A sodium dodecyl sulfate (SDS)-based inactivation method and Holder pasteurization had no effect on measures of microbial richness, while two Buffer AVL-based inactivation methods resulted in a decrease in detected richness. SDS inactivation, Holder pasteurization, and the AVL-based inactivation methods had no effect on measures of alpha diversity within samples or beta diversity between samples. Fecal DNA extracted with TRIzol-treated samples failed to amplify and sequence, making it unsuitable for microbiome analysis. These results provide guidance in the 16S rRNA microbiome analysis of fecal samples requiring viral inactivation.

scholarly journals 16S rRNA Gene Amplicon Profiling of the New Zealand parasitic blowfly Calliphora vicina

2021 ◽  
Author(s):  
Nikola Palevich ◽  
Paul H. Maclean ◽  
Luis Carvalho ◽  
Ruy Jauregui
Keyword(s):  
New Zealand ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Diversity ◽  
Adult Male ◽  
Sequence Data ◽  
Calliphora Vicina ◽  
Rrna Gene ◽  
Data Set ◽  
Male And Female

ABSTRACTHere, we present a 16S rRNA gene amplicon sequence data set and profiles demonstrating the bacterial diversity of larval and adult Calliphora vicina, collected from Ashhurst, New Zealand (May 2020). The three dominant genera among adult male and female C. vicina were Serratia and Morganella (phylum Proteobacteria) and Carnobacterium (phylum Firmicutes), while the larvae were also dominated by the genera Lactobacillus (phylum Firmicutes).

scholarly journals Effect of Bovine MFGM and LF in Infant Formula on Gut Microbiome and Metabolome at Four Months of Age

2021 ◽  
Author(s):  
Maciej Chichlowski ◽  
Nicholas Bokulich ◽  
Cheryl L Harris ◽  
Jennifer L Wampler ◽  
Fei Li ◽  
...  
Keyword(s):  
Infant Formula ◽  
Beta Diversity ◽  
Milk Fat ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Term Infants ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Metabolite Profiles

Abstract Background Milk fat globule membrane (MFGM) and lactoferrin (LF) are human milk bioactive components demonstrated to support gastrointestinal (GI) and immune development. Significantly fewer diarrhea and respiratory-associated adverse events through 18 months of age were previously reported in healthy term infants fed a cow's milk-based infant formula with added source of bovine MFGM and bovine LF through 12 months of age. Objectives To compare microbiota and metabolite profiles in a subset of study participants. Methods Stool samples were collected at Baseline (10–14 days of age) and Day 120 (MFGM + LF: 26, Control: 33). Bacterial community profiling was performed via16S rRNA gene sequencing (Illumina MiSeq) and alpha and beta diversity were analyzed (QIIME 2). Differentially abundant taxa were determined using Linear discriminant analysis effect size (LefSE) and visualized (Metacoder). Untargeted stool metabolites were analyzed (HPLC/mass spectroscopy) and expressed as the fold-change between group means (Control: MFGM + LF ratio). Results Alpha diversity increased significantly in both groups from baseline to 4 months. Subtle group differences in beta diversity were demonstrated at 4 months (Jaccard distance; R2 = 0.01, P = 0.042). Specifically, Bacteroides uniformis and Bacteroides plebeius were more abundant in the MFGM + LF group at 4 months. Metabolite profile differences for MFGM + LF vs Control included: lower fecal medium chain fatty acids, deoxycarnitine, and glycochenodeoxycholate, and some higher fecal carbohydrates and steroids (P < 0.05). After applying multiple test correction, the differences in stool metabolomics were not significant. Conclusions Addition of bovine MFGM and LF in infant formula was associated with subtle differences in stool microbiome and metabolome by four months of age, including increased prevalence of Bacteroides species. Stool metabolite profiles may be consistent with altered microbial metabolism. Trial registration:  https://clinicaltrials.gov/ct2/show/NCT02274883).

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

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

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

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

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 &lt; 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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