scholarly journals Doing More with Less: A Comparison of 16S Hypervariable Regions in Search of Defining the Shrimp Microbiota

2020 ◽  
Vol 8 (1) ◽  
pp. 134 ◽  
Author(s):  
Rodrigo García-López ◽  
Fernanda Cornejo-Granados ◽  
Alonso A. Lopez-Zavala ◽  
Filiberto Sánchez-López ◽  
Andrés Cota-Huízar ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Low Cost ◽  
Hypervariable Region ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Hypervariable Regions ◽  
Reference Databases ◽  
Marine Product ◽  
V3 Region

The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

scholarly journals Bioconductor workflow for microbiome data analysis: from raw reads to community analyses

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1492 ◽  
Author(s):  
Ben J. Callahan ◽  
Kris Sankaran ◽  
Julia A. Fukuyama ◽  
Paul J. McMurdie ◽  
Susan P. Holmes
Keyword(s):  
High Throughput Sequencing ◽  
Linear Models ◽  
Reference Database ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Nonparametric Testing ◽  
Abundance Estimates ◽  
Taxonomic Markers ◽  
Microbiome Data ◽  
Microbiome Data Analysis

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package.

scholarly journals Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA

2021 ◽  
Vol 9 (8) ◽  
pp. 1721
Author(s):  
Christian O’Dea ◽  
Roger Huerlimann ◽  
Nicole Masters ◽  
Anna Kuballa ◽  
Cameron Veal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Microbial Diversity ◽  
Surface Waters ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Faecal Contamination ◽  
Hypervariable Regions ◽  
Geographical Regions ◽  
V3 Region

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals’ faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1–V3, V3–V4, and V5–V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3–V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3–V4 (n = 11) and V5–V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

scholarly journals Microbial Composition of Human Appendices from Patients following Appendectomy

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Caitriona M. Guinane ◽  
Amany Tadrous ◽  
Fiona Fouhy ◽  
C. Anthony Ryan ◽  
Eugene M. Dempsey ◽  
...  
Keyword(s):  
Human Health ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Evolutionary Development ◽  
Rrna Gene ◽  
Limited Data ◽  
Microbial Composition ◽  
Content Type ◽  
Study Results ◽  
Human Appendix

ABSTRACT The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health. IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

scholarly journals The Equine Faecal Microbiota Undergoes Step-wise Compositional Changes in Accordance With Increasing Levels of Domestication

2021 ◽  
Author(s):  
Katie Bull ◽  
Gareth Davies ◽  
Timothy Patrick Jenkins ◽  
Laura Elizabeth Peachey
Keyword(s):  
Adult Female ◽  
High Throughput Sequencing ◽  
Rrna Gene ◽  
Faecal Microbiota ◽  
Microbial Composition ◽  
Faecal Samples ◽  
High Incidence ◽  
Life Threatening ◽  
Group Variation ◽  
The Uk

Abstract BackgroundChanges to the gut microbiota are associated with an increased incidence of disease in many species. This is particularly important during the process of domestication, where captive animals commonly suffer from gastrointestinal (GI) pathology. Horses are a prime example of a species which suffers from a high incidence of (often life-threatening) GI diseases in domesticated environments. We aimed to indentify the gut microbial changes which occur due to domestication in horses by profiling the faecal microbiota of adult female Exmoor ponies under three management conditions, representing increasing levels of domestication.MethodsFaecal samples were collected from 29 adult female Exmoor ponies in the South West of the UK; ponies were categorised as Feral (n=10), Semi-Feral (n=10) and Domesticated (n=9), based on their management conditions; thus controlling for age, gender and random effects between groups. Diet and medication were recorded and faecal samples taken to assess parasite infection. Faecal microbial composition was profiled via high-throughput sequencing of the bacterial 16S rRNA gene.ResultsDownstream biostatistical analysis indicated profound step-wise changes in global microbial community structure in the transition from Feral to Semi-Feral to Domesticated groups. A relatively high abundance of members of the phylum Proteobacteria and Tenericutes were associated with the Domesticated group; and higher levels of Methanobacteria were seen in the Feral group. The Semi-Feral group frequently had intermediate levels of these taxa; however, they also exhibited the greatest ‘within group’ variation in bacterial diversity and parasites burdens. Functional predictions revealed increased amino acid and lipid metabolism in the Domesticated group and increased energy metabolism in the Feral group; supporting a hypothesis that differences in diet was the key driver of gut microbial composition. ConclusionsIf assumed the Feral population has a more natural gut microbial phenotype, akin to that with which horses have evolved, these data can potentially be used to provide microbial signitures of balanced gut homeostasis in horses; which, in turn, will aid prevention of GI disease in domesticated horses.

scholarly journals Characterization of the salivary microbiome in people with obesity

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4458 ◽  
Author(s):  
Yujia Wu ◽  
Xiaopei Chi ◽  
Qian Zhang ◽  
Feng Chen ◽  
Xuliang Deng
Keyword(s):  
High Throughput Sequencing ◽  
Periodontal Diseases ◽  
Illumina Miseq ◽  
Normal Weight ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Oral Diseases ◽  
And Function ◽  
Healthy Participants

Background The interactions between the gut microbiome and obesity have been extensively studied. Although the oral cavity is the gateway to the gut, and is extensively colonized with microbes, little is known about the oral microbiome in people with obesity. In the present study, we investigated the salivary microbiome in obese and normal weight healthy participants using metagenomic analysis. The subjects were categorized into two groups, obesity and normal weight, based on their BMIs. Methods We characterized the salivary microbiome of 33 adults with obesity and 29 normal weight controls using high-throughput sequencing of the V3–V4 region of the 16S rRNA gene (Illumina MiSeq). None of the selected participants had systemic, oral mucosal, or periodontal diseases. Results The salivary microbiome of the obesity group was distinct from that of the normal weight group. The salivary microbiome of periodontally healthy people with obesity had both significantly lower bacterial diversity and richness compared with the controls. The genus Prevotella, Granulicatella, Peptostreptococcus, Solobacterium, Catonella, and Mogibacterium were significantly more abundant in the obesity group; meanwhile the genus Haemophilus, Corynebacterium, Capnocytophaga, and Staphylococcus were less abundant in the obesity group. We also performed a functional analysis of the inferred metagenomes, and showed that the salivary community associated with obesity had a stronger signature of immune disease and a decreased functional signature related to environmental adaptation and Xenobiotics biodegradation compared with the normal weight controls. Discussion Our study demonstrates that the microbial diversity and structure of the salivary microbiome in people with obesity are significantly different from those of normal weight controls. These results suggested that changes in the structure and function of salivary microbiome in people with obesity might reflect their susceptibility to oral diseases.

scholarly journals Characterization of Shallow Whole-Metagenome Shotgun Sequencing as a High-Accuracy and Low-Cost Method by Complicated Mock Microbiomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenyi Xu ◽  
Tianda Chen ◽  
Yuwei Pei ◽  
Hao Guo ◽  
Zhuanyu Li ◽  
...  
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
Shotgun Sequencing ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Cost Efficient ◽  
Taxonomic Assignments

Characterization of the bacterial composition and functional repertoires of microbiome samples is the most common application of metagenomics. Although deep whole-metagenome shotgun sequencing (WMS) provides high taxonomic resolution, it is generally cost-prohibitive for large longitudinal investigations. Until now, 16S rRNA gene amplicon sequencing (16S) has been the most widely used approach and usually cooperates with WMS to achieve cost-efficiency. However, the accuracy of 16S results and its consistency with WMS data have not been fully elaborated, especially by complicated microbiomes with defined compositional information. Here, we constructed two complex artificial microbiomes, which comprised more than 60 human gut bacterial species with even or varied abundance. Utilizing real fecal samples and mock communities, we provided solid evidence demonstrating that 16S results were of poor consistency with WMS data, and its accuracy was not satisfactory. In contrast, shallow whole-metagenome shotgun sequencing (shallow WMS, S-WMS) with a sequencing depth of 1 Gb provided outputs that highly resembled WMS data at both genus and species levels and presented much higher accuracy taxonomic assignments and functional predictions than 16S, thereby representing a better and cost-efficient alternative to 16S for large-scale microbiome studies.

scholarly journals Systematic processing of ribosomal RNA gene amplicon sequencing data

GigaScience ◽  
2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Julien Tremblay ◽  
Etienne Yergeau
Keyword(s):  
Ribosomal Rna ◽  
High Performance ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Low Cost ◽  
Marker Gene ◽  
Fine Tuning ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Data Types

Abstract Background With the advent of high-throughput sequencing, microbiology is becoming increasingly data-intensive. Because of its low cost, robust databases, and established bioinformatic workflows, sequencing of 16S/18S/ITS ribosomal RNA (rRNA) gene amplicons, which provides a marker of choice for phylogenetic studies, has become ubiquitous. Many established end-to-end bioinformatic pipelines are available to perform short amplicon sequence data analysis. These pipelines suit a general audience, but few options exist for more specialized users who are experienced in code scripting, Linux-based systems, and high-performance computing (HPC) environments. For such an audience, existing pipelines can be limiting to fully leverage modern HPC capabilities and perform tweaking and optimization operations. Moreover, a wealth of stand-alone software packages that perform specific targeted bioinformatic tasks are increasingly accessible, and finding a way to easily integrate these applications in a pipeline is critical to the evolution of bioinformatic methodologies. Results Here we describe AmpliconTagger, a short rRNA marker gene amplicon pipeline coded in a Python framework that enables fine tuning and integration of virtually any potential rRNA gene amplicon bioinformatic procedure. It is designed to work within an HPC environment, supporting a complex network of job dependencies with a smart-restart mechanism in case of job failure or parameter modifications. As proof of concept, we present end results obtained with AmpliconTagger using 16S, 18S, ITS rRNA short gene amplicons and Pacific Biosciences long-read amplicon data types as input. Conclusions Using a selection of published algorithms for generating operational taxonomic units and amplicon sequence variants and for computing downstream taxonomic summaries and diversity metrics, we demonstrate the performance and versatility of our pipeline for systematic analyses of amplicon sequence data.

scholarly journals A novel PCR-clamping assay reducing plant host DNA amplification significantly improves prokaryotic endo-microbiome community characterization

2020 ◽  
Vol 96 (7) ◽  
Author(s):  
Emilie Lefèvre ◽  
Courtney M Gardner ◽  
Claudia K Gunsch
Keyword(s):  
Mitochondrial Dna ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Dna Amplification ◽  
Rrna Gene ◽  
Plant Host ◽  
Plant Chloroplast

ABSTRACT Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers.

scholarly journals Benchmarking DNA isolation kits used in analyses of the urinary microbiome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa Karstens ◽  
Nazema Y. Siddiqui ◽  
Tamara Zaza ◽  
Alecsander Barstad ◽  
Cindy L. Amundsen ◽  
...  
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Dna Isolation ◽  
Urine Samples ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Alpha And Beta Diversity ◽  
Dna Yield ◽  
Urinary Microbiome ◽  
Buffer Control

AbstractThe urinary microbiome has been increasingly characterized using next-generation sequencing. However, many of the technical methods have not yet been specifically optimized for urine. We sought to compare the performance of several DNA isolation kits used in urinary microbiome studies. A total of 11 voided urine samples and one buffer control were divided into 5 equal aliquots and processed in parallel using five commercial DNA isolation kits. DNA was quantified and the V4 segment of the 16S rRNA gene was sequenced. Data were processed to identify the microbial composition and to assess alpha and beta diversity of the samples. Tested DNA isolation kits result in significantly different DNA yields from urine samples. DNA extracted with the Qiagen Biostic Bacteremia and DNeasy Blood & Tissue kits showed the fewest technical issues in downstream analyses, with the DNeasy Blood & Tissue kit also demonstrating the highest DNA yield. Nevertheless, all five kits provided good quality DNA for high throughput sequencing with non-significant differences in the number of reads recovered, alpha, or beta diversity.

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