scholarly journals An updated genetic marker for detection of Lake Sinai Virus and metagenetic applications

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9424
Author(s):  
Deborah D. Iwanowicz ◽  
Judy Y. Wu-Smart ◽  
Tugce Olgun ◽  
Autumn H. Smart ◽  
Clint R.V. Otto ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Compositional Analysis ◽  
Illumina Miseq ◽  
Similar Rate ◽  
End Point ◽  
Distinct Variable ◽  
Paired Samples ◽  
Sequencing Library ◽  
Halictus Ligatus ◽  
Sample Set

Background Lake Sinai Viruses (LSV) are common RNA viruses of honey bees (Apis mellifera) that frequently reach high abundance but are not linked to overt disease. LSVs are genetically heterogeneous and collectively widespread, but despite frequent detection in surveys, the ecological and geographic factors structuring their distribution in A. mellifera are not understood. Even less is known about their distribution in other species. Better understanding of LSV prevalence and ecology have been hampered by high sequence diversity within the LSV clade. Methods Here we report a new polymerase chain reaction (PCR) assay that is compatible with currently known lineages with minimal primer degeneracy, producing an expected 365 bp amplicon suitable for end-point PCR and metagenetic sequencing. Using the Illumina MiSeq platform, we performed pilot metagenetic assessments of three sample sets, each representing a distinct variable that might structure LSV diversity (geography, tissue, and species). Results The first sample set in our pilot assessment compared cDNA pools from managed A. mellifera hives in California (n = 8) and Maryland (n = 6) that had previously been evaluated for LSV2, confirming that the primers co-amplify divergent lineages in real-world samples. The second sample set included cDNA pools derived from different tissues (thorax vs. abdomen, n = 24 paired samples), collected from managed A. mellifera hives in North Dakota. End-point detection of LSV frequently differed between the two tissue types; LSV metagenetic composition was similar in one pair of sequenced samples but divergent in a second pair. Overall, LSV1 and intermediate lineages were common in these samples whereas variants clustering with LSV2 were rare. The third sample set included cDNA from individual pollinator specimens collected from diverse landscapes in the vicinity of Lincoln, Nebraska. We detected LSV in the bee Halictus ligatus (four of 63 specimens tested, 6.3%) at a similar rate as A. mellifera (nine of 115 specimens, 7.8%), but only one H. ligatus sequencing library yielded sufficient data for compositional analysis. Sequenced samples often contained multiple divergent LSV lineages, including individual specimens. While these studies were exploratory rather than statistically powerful tests of hypotheses, they illustrate the utility of high-throughput sequencing for understanding LSV transmission within and among species.

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 Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

2020 ◽  
Author(s):  
Philipp Schwabl ◽  
Jalil Maiguashca Sánchez ◽  
Jaime A. Costales ◽  
Sofía Ocaña ◽  
Maikell Segovia ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Ex Vivo ◽  
Cost Effective ◽  
Illumina Miseq ◽  
Epidemiological Surveillance ◽  
Method Performance ◽  
Genome Wide ◽  
Target Dna ◽  
Polymerase Chain ◽  
Sample Set

AbstractAnalysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from vector/host material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Approximately 100 libraries can be sequenced together in one Illumina MiSeq run. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate DTUs. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

scholarly journals Evaluation of bacterial diversity during fermentation process: a comparison between handmade and machine-made high-temperature Daqu of Maotai-flavor liquor

2020 ◽  
Vol 70 (1) ◽  
Author(s):  
Qiancheng Zuo ◽  
Yongguang Huang ◽  
MinGuo
Keyword(s):  
High Temperature ◽  
Bacterial Diversity ◽  
Relative Abundance ◽  
Fermentation Process ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Functional Bacteria ◽  
Significant Difference ◽  
Fermentation Technology ◽  
Molding Methods

Abstract Purpose High-temperature Daqu is a traditional fermentation starter that is used for Chinese Maotai-flavor Baijiu production. Although the bacteria in high-temperature Daqu are known to be responsible for developing the quality and flavor of Baijiu during the fermentation process, there is little information on the properties of the bacteria during the fermentation of high-temperature Daqu, especially machine-made high-temperature Daqu. This has limited the development of the Maotai-flavor Baijiu industry, particularly with regard to the mechanized production of Maotai-flavor Baijiu. Methods Illumina MiSeq high-throughput sequencing was applied to study bacterial compositions during the fermentation of handmade and machine-made high temperatures. Results The results show that bacterial diversity in machine-made Daqu was similar but higher than that in handmade Daqu at the end of fermentation, and there was no significant difference between the methods with regard to the dominant genera and their dynamic changes during fermentation. Rhizobium, Bacillus, Thermoactinomyces, Weissella, Lactobacillus, and Saccharopolyspora were the dominant genera during the fermentation of both Daqus, although the relative abundance of these dominant genera differed between the two methods. Interestingly, the machine-made Daqu contained a higher relative abundance of Bacillus than handmade Daqu at all fermentation times. Bacillus is the most important functional bacteria in the fermentation of Maotai-flavor Baijiu, suggesting that mechanical-molding methods could be applied to industrial Maotai-flavor Daqu production. Conclusion These results suggest that mechanical-molding methods could be applied to industrial Maotai-flavor Daqu production, which could be helpful for industrial Maotai-flavor Baijiu production and the development of fermentation technology.

Analysis of the effects of nanosilver on bacterial community in the intestinal fluid of silkworms using high-throughput sequencing

2019 ◽  
Vol 110 (3) ◽  
pp. 309-320
Author(s):  
Chen Lin ◽  
Zhou Wei ◽  
Zhou Yi ◽  
Tan Tingting ◽  
Du Huamao ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Intestinal Bacteria ◽  
Illumina Miseq ◽  
The Body ◽  
Dependent Manner ◽  
Negative Effects ◽  
Silkworm Larvae ◽  
Intestinal Bacterial Community

AbstractNanosilver is an environment-friendly, harmless alternative of traditional disinfectants which can be potentially applied in the sericulture industry. However, the effects of nanosilver on the intestinal bacterial community of the silkworms (Bombyx mori L.) are unclear. In this study, Illumina MiSeq high-throughput sequencing technology was used to assess the intestinal bacterial community in both male and female silkworms while treated with different concentrations of nanosilver. We found that nanosilver significantly influenced the composition of silkworm intestinal bacterial community on the different taxonomic levels. Most conspicuously, the abundance of Firmicutes was increased by the treatment of 20 mg L−1 nanosilver but decreased by that of 100 mg L−1 nanosilver at the phylum level. The same trend was observed in Bacilli at the class level and in Enterococcus at the genus level. In some extreme cases, application of nanosilver eliminated the bacterium, e.g., Brevibacillus, but increased the population of several other bacteria in the host intestine, such as Blautia, Terrisporobacter, Faecalibacterium, and some bacteria could only be found in nanosilver treatment groups, e.g., Dialister. In addition, although nanosilver generally showed negative effects on the cocooning rate in a dose-dependent manner, we found that 20 mg L−1 nanosilver treatment significantly increased the body weight of silkworms and did not show negative effects on the survival rate. These results indicated that the intestinal bacteria community of silkworm larvae was significantly changed after nanosilver treatment which might consequently influence host growth and development.

scholarly journals DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

2015 ◽  
Vol 105 (6) ◽  
pp. 717-727 ◽  
Author(s):  
G.-J. Brandon-Mong ◽  
H.-M. Gan ◽  
K.-W. Sing ◽  
P.-S. Lee ◽  
P.-E. Lim ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Ease Of Use ◽  
Malaise Trap ◽  
Detection Rates ◽  
Operational Taxonomic Units ◽  
Arthropod Species ◽  
Primer Sets ◽  
Arthropod Biodiversity

AbstractMetabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

scholarly journals Small RNA Expression Profiling by High-Throughput Sequencing: Implications of Enzymatic Manipulation

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Fanglei Zhuang ◽  
Ryan T. Fuchs ◽  
G. Brett Robb
Keyword(s):  
High Throughput ◽  
Expression Profiling ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Biological Processes ◽  
Cellular Processes ◽  
Disease States ◽  
Powerful Approach ◽  
Sequencing Library ◽  
Rna Expression Profiling

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

scholarly journals Distribution of Hydrogen-Producing Bacteria in Tibetan Hot Springs, China

2021 ◽  
Vol 12 ◽  
Author(s):  
Li Ma ◽  
Geng Wu ◽  
Jian Yang ◽  
Liuqin Huang ◽  
Dorji Phurbu ◽  
...  
Keyword(s):  
Community Structure ◽  
Hydrogen Production ◽  
16S Rrna ◽  
High Throughput Sequencing ◽  
Hot Springs ◽  
Illumina Miseq ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
The Tibetan Plateau

Investigating the distribution of hydrogen-producing bacteria (HPB) is of great significance to understanding the source of biological hydrogen production in geothermal environments. Here, we explored the compositions of HPB populations in the sediments of hot springs from the Daggyai, Quzhuomu, Quseyongba, and Moluojiang geothermal zones on the Tibetan Plateau, with the use of Illumina MiSeq high-throughput sequencing of 16S rRNA genes and hydA genes. In the present study, the hydA genes were successfully amplified from the hot springs with a temperature of 46–87°C. The hydA gene phylogenetic analysis showed that the top three phyla of the HPB populations were Bacteroidetes (14.48%), Spirochaetes (14.12%), and Thermotogae (10.45%), while Proteobacteria were absent in the top 10 of the HPB populations, although Proteobacteria were dominant in the 16S rRNA gene sequences. Canonical correspondence analysis results indicate that the HPB community structure in the studied Tibetan hot springs was correlated with various environmental factors, such as temperature, pH, and elevation. The HPB community structure also showed a spatial distribution pattern; samples from the same area showed similar community structures. Furthermore, one HPB isolate affiliated with Firmicutes was obtained and demonstrated the capacity of hydrogen production. These results are important for us to understand the distribution and function of HPB in hot springs.

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 Micro-community associated with ectomycorrhizal Russula symbiosis and sporocarp-producing Russula in Fagaceae dominant nature areas in southern China

2020 ◽  
Author(s):  
Wen Ying Yu ◽  
Ming Hui Peng ◽  
Jia Jia Wang ◽  
Wen Yu Ye ◽  
Zong Hua Wang ◽  
...  
Keyword(s):  
Indicator Species ◽  
High Throughput Sequencing ◽  
Southern China ◽  
Illumina Miseq ◽  
Soil Microbiome ◽  
Root Tips ◽  
Network Analyses ◽  
Mycorrhizal Interactions ◽  
Type 3

ABSTRACTRussula griseocarnosa, an ectomycorrhizal (ECM) fungus, is a species of precious wild edible mushrooms with very high market value in southern China. Its yield is affected by many factors including the tree species and environmental conditions such as soil microbiome, humidity. How the microbiome promotes the ECM fungus symbiosis with Fagaceae plants and sporocarp-producing has never been studied. In this study, we collected rhizosphere samples from Fujian province, the microbiota in the root and mycorrhizal rhizosphere were identified by Illumina MiSeq high-throughput sequencing. First, we compared three types of fungal communities: root tips infected with ECM Russula (type 1), tips with Russula sporocarp (type 2) and tips without ECM (type 3). Our results showed that the fungal richness was negatively correlated with Russula. Russula, Tomentella and Lactarius were common in Fagaceae ECM roots. As to the mycorrhizal interactions, Boletus may be considered as an indicator species for sporocarp-producing Russula, and Acremonium, Cladophialophora were associated with Russula symbiosis. Second, we analyzed the fungal and bacterial communities in rhizosphere soils from the corresponding to previously three types (type 1, 2, 3). Dacryobolus and Acidocella may be considered as an indicator species for sporocarp-producing Russula. Fungi Tomentella, Saitozyma, Elaphomyces and bacteria Acidicaldus, Bryobacter, Sorangium and Acidobacterium occurred more frequently in the ECM Russula rhizosphere. Furthermore, the indicators Elaphomyces, Tomentella, Sorangium had a positive correlation with Russula symbiosis by network analyses. Overall, our results suggest a relationship between micro-community and ECM Russula formation and Russula sporocarp, which may provide new strategies for improving Russula symbiosis rate and sporocarp production.

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