scholarly journals Assessment of Bifidobacterium Species Using groEL Gene on the Basis of Illumina MiSeq High-Throughput Sequencing

Genes ◽  
2017 ◽  
Vol 8 (11) ◽  
pp. 336 ◽  
Author(s):  
Lujun Hu ◽  
Wenwei Lu ◽  
Linlin Wang ◽  
Mingluo Pan ◽  
Hao Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Groel Gene ◽  
Bifidobacterium Species

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.

Diversity of Entamoeba spp. in African great apes and humans: an insight from Illumina MiSeq high-throughput sequencing

2018 ◽  
Vol 48 (7) ◽  
pp. 519-530 ◽  
Author(s):  
Klára Vlčková ◽  
Jakub Kreisinger ◽  
Barbora Pafčo ◽  
Dagmar Čížková ◽  
Nikki Tagg ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Great Apes ◽  
African Great Apes

scholarly journals Rapid isolation and characterization of microsatellites in the critically endangered Mountain Bongo (Tragelaphus eurycerus isaaci)

2019 ◽  
Author(s):  
Fraser John Combe ◽  
Evelyn Taylor cox ◽  
Graeme fox ◽  
Tommy Sandri ◽  
Bradley Cain ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Population Analysis ◽  
Illumina Miseq ◽  
Model Organisms ◽  
Conservation Genomics ◽  
Adaptive Genetic Variation ◽  
Genetics Research ◽  
Isolation And Characterization ◽  
Neutral Markers

High-throughput sequencing tools promise to revolutionize many aspects of genetics research, e.g. by allowing the identification of functional adaptive genetic variation. However, the expense and expertise required to apply these tools to basic conservation questions is a challenge for applications outside academia, resulting in a so-called “conservation genomics gap” (Shafer et al. 2015). The conservation genetics paradigm is that basic information about inbreeding and gene flow are often critical to inform conservation management of small populations (Ouborg et al. 2010). This information is often needed quickly and ideally should be accessible to workers without special expertise in genomics (DeSalle & Amato 2004). While the inferential power of high-throughput sequencing to interrogate the genome is profound, the cost for population analysis is higher (though decreasing) than for traditional neutral markers. Thus, the use of neutral markers is still relevant in conservation applications. However, this assumes that neutral markers have been discovered and characterized for a given species of conservation concern, which is often untrue for non-model organisms. Here, we use a fast, cost-efficient, high-throughput sequencing method (Illumina MiSeq) to rapidly identify and characterize microsatellites in the mountain bongo (Tragelaphus eurycerus isaaci, hereafter bongo), which has a clear and timely conservation imperative but lacks any described neutral markers.

scholarly journals Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2

2021 ◽  
Author(s):  
Marc Fuchs ◽  
Clara Radulescu ◽  
Miao Tang ◽  
Arun Mahesh ◽  
Deborah Lavin ◽  
...  
Keyword(s):  
High Throughput ◽  
Phylogenetic Trees ◽  
High Throughput Sequencing ◽  
Cost Savings ◽  
Cost Effective ◽  
Illumina Miseq ◽  
Pass Rate ◽  
Library Preparation ◽  
Reaction Volumes ◽  
Library Preparation Protocol

Introduction: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost one million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. Methods: SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. Results: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io (https://dx.doi.org/10.17504/protocols.io.bvntn5en). The final version of Mini-XT has been used to sequence 4,384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. Conclusion: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes 8-fold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

scholarly journals The Fungal Communities and Flavor Profiles in Different Types of High-Temperature Daqu as Revealed by High-Throughput Sequencing and Electronic Senses

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenchao Cai ◽  
Yu’ang Xue ◽  
Yurong Wang ◽  
Wenping Wang ◽  
Na Shu ◽  
...  
Keyword(s):  
High Temperature ◽  
Nitrogen Oxides ◽  
Organic Compounds ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Electronic Tongue ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Significant Difference ◽  
Distinct Character

Polymicrobial co-fermentation is among the distinct character of high-temperature Daqu. However, fungal communities in the three types of high-temperature Daqu, namely, white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, are yet to be characterized. In this study, the fungal diversity, taste, and aroma profiles in the three types of high-temperature Daqu were investigated by Illumina MiSeq high-throughput sequencing, electronic tongue, and electronic nose, respectively. Ascomycota and Basidiomycota were detected as the absolute dominant fungal phylum in all types of high-temperature Daqu samples, whereas Thermomyces, Thermoascus, Aspergillus, Rasamsonia, Byssochlamys, and Trichomonascus were identified as the dominant fungal genera. The fungal communities of the three types of high-temperature Daqu differed significantly (p < 0.05), and Thermomyces, Thermoascus, and Monascus could serve as the biomarkers in white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, respectively. The three types of high-temperature Daqu had an extremely significant difference (p < 0.01) in flavor: white high-temperature Daqu was characterized by sourness, bitterness, astringency, richness, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds; black high-temperature Daqu was characterized by aftertaste-A, aftertaste-B, methane-aliph, hydrogen, and aromatic compounds; and yellow high-temperature Daqu was characterized by saltiness, umami, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds. The fungal communities in the three types of high-temperature Daqu were significantly correlated with taste but not with aroma, and the aroma of high-temperature Daqu was mainly influenced by the dominant fungal genera including Trichomonascus, Aspergillus, Thermoascus, and Thermomyces. The result of the present study enriched and refined our knowledge of high-temperature Daqu, which had positive implications for the development of traditional brewing technique.

scholarly journals Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

2018 ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

AbstractAmplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.ImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

scholarly journals groEL Gene-Based Phylogenetic Analysis of Lactobacillus Species by High-Throughput Sequencing

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 530 ◽  
Author(s):  
Xie ◽  
Pan ◽  
Jiang ◽  
Liu ◽  
Lu ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Hypervariable Region ◽  
Single Copy ◽  
Species Level ◽  
Gene Profiling ◽  
Lactobacillus Species ◽  
Groel Gene ◽  
Lactobacillus Mucosae

Lactobacillus is a fairly diverse genus of bacteria with more than 260 species and subspecies. Many profiling methods have been developed to carry out phylogenetic analysis of this complex and diverse genus, but limitations remain since there is still a lack of comprehensive and accurate analytical method to profile this genus at species level. To overcome these limitations, a Lactobacillus-specific primer set was developed targeting a hypervariable region in the groEL gene—a single-copy gene that has undergone rapid mutation and evolution. The results showed that this methodology could accurately perform taxonomic identification of Lactobacillus down to the species level. Its detection limit was as low as 104 colony-forming units (cfu)/mL for Lactobacillus species. The assessment of detection specificity using the Lactobacillus groEL profiling method found that Lactobacillus, Pediococcus, Weissella, and Leuconostoc genus could be distinguished, but non-Lactobacillus Genus Complex could not be detected. The groEL gene sequencing and Miseq high-throughput approach were adopted to estimate the richness and diversity of Lactobacillus species in different ecosystems. The method was tested using kurut (fermented yak milk) samples and fecal samples of human, rat, and mouse. The results indicated that Lactobacillus mucosae was the predominant gut Lactobacillus species among Chinese, and L. johnsonii accounted for the majority of lactobacilli in rat and mouse gut. Meanwhile, L. delbrueckii subsp. bulgaricus had the highest relative abundance of Lactobacillus in kurut. Thus, this groEL gene profiling method is expected to promote the application of Lactobacillus for industrial production and therapeutic purpose.

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