scholarly journals Consequences of organ choice in describing bacterial pathogen assemblages in a rodent population

2017 ◽  
Vol 145 (14) ◽  
pp. 3070-3075 ◽  
Author(s):  
P. VILLETTE ◽  
E. AFONSO ◽  
G. COUVAL ◽  
A. LEVRET ◽  
M. GALAN ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bacterial Pathogens ◽  
Bacterial Pathogen ◽  
Cost Effective ◽  
Operational Taxonomic Unit ◽  
Amplicon Sequencing ◽  
Bacterial Assemblage ◽  
Sequencing Technologies ◽  
16S Rrna Amplicon Sequencing ◽  
Bacterial Richness

SUMMARYHigh-throughput sequencing technologies now allow for rapid cost-effective surveys of multiple pathogens in many host species including rodents, but it is currently unclear if the organ chosen for screening influences the number and identity of bacteria detected. We used 16S rRNA amplicon sequencing to identify bacterial pathogens in the heart, liver, lungs, kidneys and spleen of 13 water voles (Arvicola terrestris) collected in Franche-Comté, France. We asked if bacterial pathogen assemblages within organs are similar and if all five organs are necessary to detect all of the bacteria present in an individual animal. We identified 24 bacteria representing 17 genera; average bacterial richness for each organ ranged from 1·5 ± 0·4 (mean ± standard error) to 2·5 ± 0·4 bacteria/organ and did not differ significantly between organs. The average bacterial richness when organ assemblages were pooled within animals was 4·7 ± 0·6 bacteria/animal; Operational Taxonomic Unit accumulation analysis indicates that all five organs are required to obtain this. Organ type influences bacterial assemblage composition in a systematic way (PERMANOVA, 999 permutations, pseudo-F4,51 = 1·37, P = 0·001). Our results demonstrate that the number of organs sampled influences the ability to detect bacterial pathogens, which can inform sampling decisions in public health and wildlife ecology.

scholarly journals Combining whole genome shotgun sequencing and rDNA amplicon analyses to improve detection of microbe-microbe interaction networks in plant leaves

2019 ◽  
Author(s):  
Julian Regalado ◽  
Derek S. Lundberg ◽  
Oliver Deusch ◽  
Sonja Kersten ◽  
Talia Karasov ◽  
...  
Keyword(s):  
16S Rdna ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Low Complexity ◽  
Shotgun Sequencing ◽  
Wild Plants ◽  
Hybrid Strategy ◽  
Domains Of Life ◽  
Metagenome Sequencing

AbstractMicroorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, acquire nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of 16S rDNA and/or the internal transcribed spacer (ITS) of rDNA loci, but the decreasing cost of high-throughput sequencing has made shotgun metagenome sequencing increasingly accessible. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S rDNA and eukaryotic ITS1 amplicon data from 176 of these samples. The shotgun data were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. For shotgun and amplicon data, microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. We use the metagenome data, which captures the ratio of bacterial to plant DNA in leaves of wild plants, to scale the 16S rDNA amplicon data such that they reflect absolute bacterial abundance. We show that this cost-effective hybrid strategy overcomes compositionality problems in amplicon data and leads to fundamentally different conclusions about microbiome community assembly.

scholarly journals Synthetic Sequencing Standards: A Guide to Database Choice for Rumen Microbiota Amplicon Sequencing Analysis

2020 ◽  
Vol 11 ◽  
Author(s):  
Paul E. Smith ◽  
Sinead M. Waters ◽  
Ruth Gómez Expósito ◽  
Hauke Smidt ◽  
Ciara A. Carberry ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Gas Production ◽  
Reference Database ◽  
Specific Reference ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Rumen Microbiota ◽  
Reference Databases

Our understanding of complex microbial communities, such as those residing in the rumen, has drastically advanced through the use of high throughput sequencing (HTS) technologies. Indeed, with the use of barcoded amplicon sequencing, it is now cost effective and computationally feasible to identify individual rumen microbial genera associated with ruminant livestock nutrition, genetics, performance and greenhouse gas production. However, across all disciplines of microbial ecology, there is currently little reporting of the use of internal controls for validating HTS results. Furthermore, there is little consensus of the most appropriate reference database for analyzing rumen microbiota amplicon sequencing data. Therefore, in this study, a synthetic rumen-specific sequencing standard was used to assess the effects of database choice on results obtained from rumen microbial amplicon sequencing. Four DADA2 reference training sets (RDP, SILVA, GTDB, and RefSeq + RDP) were compared to assess their ability to correctly classify sequences included in the rumen-specific sequencing standard. In addition, two thresholds of phylogenetic bootstrapping, 50 and 80, were applied to investigate the effect of increasing stringency. Sequence classification differences were apparent amongst the databases. For example the classification of Clostridium differed between all databases, thus highlighting the need for a consistent approach to nomenclature amongst different reference databases. It is hoped the effect of database on taxonomic classification observed in this study, will encourage research groups across various microbial disciplines to develop and routinely use their own microbiome-specific reference standard to validate analysis pipelines and database choice.

scholarly journals Natrix: A Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads

2020 ◽  
Author(s):  
Marius Welzel ◽  
Anja Lange ◽  
Dominik Heider ◽  
Michael Schwarz ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Workflow Management ◽  
Amplicon Sequencing ◽  
Version Control ◽  
Marker Genes ◽  
Sequencing Data ◽  
Taxonomic Assignment ◽  
Ecological Processes ◽  
Sequencing Technologies ◽  
User Friendly

AbstractSequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires effcient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an effcient workflow management system. We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix).

An investigation into the anaerobic spoilage microbiota of beef carcass and rump steak cuts using high- throughput sequencing

2021 ◽  
Author(s):  
Eden Esteves ◽  
Paul Whyte ◽  
John Mills ◽  
Gale Brightwell ◽  
Tanushree B Gupta ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bioinformatics Analysis ◽  
Amplicon Sequencing ◽  
Alpha And Beta Diversity ◽  
Operational Taxonomic Units ◽  
16S Rrna Amplicon Sequencing ◽  
Meat Spoilage ◽  
Beef Carcasses ◽  
Spoilage Bacteria ◽  
Beef Carcass

Abstract The presence of anaerobic microflora on fresh beef carcass and rump steaks, which may contribute to meat spoilage, was explored in this study. A total of 120 carcass and 120 rump steak swabs were collected immediately after slaughtering and boning, respectively from five meat plants, anaerobically incubated and enriched d at 4°C for 3 weeks. This was followed by DNA extraction and 16S rRNA amplicon sequencing using the Illumina MiSeqTM, with subsequent bioinformatics analysis. The enriched microbiota of the samples was classified and grouped into 149 operational taxonomic units (OTUs). The microbiota recovered from both sample types consisted mainly of Carnobacterium, with an average relative abundance of 28.4% and 32.8% in beef carcasses and beef rump steaks, respectively. This was followed by Streptococcus, Serratia, Lactococcus, Enterococcus, Escherichia-Shigella, Raoultella and Aeromonas ranging from 1.5–20% and 0.1–29.8% in enriched carcasses and rump steak swabs, respectively. Trichococcus, Bacteroides, Dysgomonas, Providencia, Paraclostridium and Proteus were also present ranging from 0–0.8% on carcass and 0–1.8% on rump steak swabs, respectively. Alpha and Beta diversity measurements showed limited diversity between the two sample types, but some differences between samples from the beef plants investigated were evident. This study highlights the presence of potential spoilage bacteria, mainly anaerobic genera on and between carcass and rump steaks, as an indication of contamination on and between these samples.

scholarly journals Instability and Stasis Among the Microbiome of Seagrass Leaves, Roots and Rhizomes, and Nearby Sediments Within a Natural pH Gradient

2021 ◽  
Author(s):  
Raymond B. Banister ◽  
Melbert T. Schwarz ◽  
Maoz Fine ◽  
Kim B. Ritchie ◽  
Erinn M. Muller
Keyword(s):  
Bacterial Community ◽  
Microbial Communities ◽  
Elevated Temperatures ◽  
Operational Taxonomic Unit ◽  
Amplicon Sequencing ◽  
Ph Gradient ◽  
Ecological Value ◽  
Seagrass Meadows ◽  
16S Rrna Amplicon Sequencing ◽  
Natural Ph Gradient

AbstractSeagrass meadows are hotspots of biodiversity with considerable economic and ecological value. The health of seagrass ecosystems is influenced in part by the makeup and stability of their microbiome, but microbiome composition can be sensitive to environmental change such as nutrient availability, elevated temperatures, and reduced pH. The objective of the present study was to characterize the bacterial community of the leaves, bulk samples of roots and rhizomes, and proximal sediment of the seagrass species Cymodocea nodosa along the natural pH gradient of Levante Bay, Vulcano Island, Italy. The bacterial community was determined by characterizing the 16S rRNA amplicon sequencing and analyzing the operational taxonomic unit classification of bacterial DNA within samples. Statistical analyses were used to explore how life-long exposure to different pH/pCO2 conditions may be associated with significant differences in microbial communities, dominant bacterial classes, and microbial diversity within each plant section and sediment. The microbiome of C. nodosa significantly differed among all sample types and site-specific differences were detected within sediment and root/rhizome microbial communities, but not the leaves. These results show that C. nodosa leaves have a consistent microbial community even across a pH range of 8.15 to 6.05. The ability for C. nodosa to regulate and maintain microbial structure may indicate a semblance of resilience within these vital ecosystems under projected changes in environmental conditions such as ocean acidification.

scholarly journals Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasemin Guenay-Greunke ◽  
David A. Bohan ◽  
Michael Traugott ◽  
Corinna Wallinger
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Sequencing Depth ◽  
Sequencing Error ◽  
Sequencing Data ◽  
Large Sample ◽  
Sequencing Errors ◽  
Plant Feeding

AbstractHigh-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

scholarly journals 16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife: the importance of cleaning post-sequencing data before estimating positivity, prevalence and co-infection

2016 ◽  
Author(s):  
Maxime Galan ◽  
Maria Razzauti ◽  
Emilie Bard ◽  
Maria Bernard ◽  
Carine Brouat ◽  
...  
Keyword(s):  
16S Rrna ◽  
Disease Transmission ◽  
Pathogenic Bacteria ◽  
High Throughput Sequencing ◽  
Rattus Rattus ◽  
Amplicon Sequencing ◽  
Natural Habitats ◽  
16S Rrna Amplicon Sequencing ◽  
Epidemiological Surveys ◽  
Zoonotic Risk

SummaryHuman impact on natural habitats is increasing the complexity of human-wildlife interfaces and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents collected from 24 villages in Senegal, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis and 221 Mastomys erythroleucus. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus and Orientia. The last five of these genera have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable for continuous pathogen surveillance in the context of disease monitoring programs.

scholarly journals A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Kimberly R. Andrews ◽  
Samuel S. Hunter ◽  
Brandi K. Torrevillas ◽  
Nora Céspedes ◽  
Sarah M. Garrison ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
High Density ◽  
Snp Genotyping ◽  
Mouse Strains ◽  
Bioinformatic Pipeline ◽  
Genotyping Assay ◽  
Sequencing Technologies ◽  
Diagnostic Snps

Abstract Background Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. “Next generation” high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose. Results We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807–819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123–139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic. Conclusions The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

scholarly journals Cracking the genetic code of our cities: researchers around the world aim to map the urban genome

2016 ◽  
Vol 37 (4) ◽  
pp. 194
Author(s):  
Sofia Ahsanuddin ◽  
Ebrahim Afshinnekoo ◽  
Christopher E. Mason
Keyword(s):  
Genetic Code ◽  
High Throughput ◽  
Light Microscope ◽  
Scientific Revolution ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
The Past ◽  
Sequencing Technologies ◽  
The World ◽  
The One

It is rare to say that one has lived through a revolution, but we are all living through one right now. High-throughput sequencing technologies have become cheaper and more cost-effective over the past decade, moving even faster than Moore’s Law for computer power (doubling every 18 months). Because sequencers are modern-day 'molecular microscopes', scientists believe that we are currently experiencing a scientific revolution similar to the one sparked by Antonie van Leeuwenhoek’s invention of the world’s first light microscope in the 17th century.

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