scholarly journals Strategy for Modular Tagged High-Throughput Amplicon Sequencing

2011 ◽  
Vol 77 (17) ◽  
pp. 6310-6312 ◽  
Author(s):  
Daniel Aguirre de Cárcer ◽  
Stuart E. Denman ◽  
Chris McSweeney ◽  
Mark Morrison
Keyword(s):  
High Throughput ◽  
Amplicon Sequencing ◽  
Modular Approach ◽  
Specific Primer ◽  
Bar Code ◽  
Pcr Product ◽  
Primer Sets

ABSTRACTThe use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.

scholarly journals Amplicon-Based High-Throughput Sequencing Method Capable of Species-Level Identification of Coagulase-Negative Staphylococci in Diverse Communities

2020 ◽  
Vol 8 (6) ◽  
pp. 897
Author(s):  
Emiel Van Reckem ◽  
Luc De Vuyst ◽  
Frédéric Leroy ◽  
Stefan Weckx
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Coagulase Negative Staphylococci ◽  
Tuf Gene ◽  
Microbial Ecosystems ◽  
Gene Database ◽  
Primer Sets ◽  
Level Identification

Coagulase-negative staphylococci (CNS) make up a diverse bacterial group, appearing in a myriad of ecosystems. To unravel the composition of staphylococcal communities in these microbial ecosystems, a reliable species-level identification is crucial. The present study aimed to design a primer set for high-throughput amplicon sequencing, amplifying a region of the tuf gene with enough discriminatory power to distinguish different CNS species. Based on 2566 tuf gene sequences present in the public European Nucleotide Archive database and saved as a custom tuf gene database in-house, three different primer sets were designed, which were able to amplify a specific region of the tuf gene for 36 strains of 18 different CNS species. In silico analysis revealed that species-level identification of closely related species was only reliable if a 100% identity cut-off was applied for matches between the amplicon sequence variants and the custom tuf gene database. From the three primer sets designed, one set (Tuf387/765) outperformed the two other primer sets for studying Staphylococcus-rich microbial communities using amplicon sequencing, as it resulted in no false positives and precise species-level identification. The method developed offers interesting potential for a rapid and robust analysis of complex staphylococcal communities in a variety of microbial ecosystems.

Detection of two Microsporidia pathogens of the European honey bee Apis Mellifera (Insecta: Apidae) in Western Siberia

2018 ◽  
Vol 63 (4) ◽  
pp. 728-732 ◽  
Author(s):  
Yuri S. Tokarev ◽  
Zimfira Y. Zinatullina ◽  
Anastasiya N. Ignatieva ◽  
Oksana N. Zhigileva ◽  
Julia M. Malysh ◽  
...  
Keyword(s):  
Honey Bee ◽  
Honey Bees ◽  
Western Siberia ◽  
Microscopic Analysis ◽  
Nosema Apis ◽  
Specific Primer ◽  
Emerging Pathogen ◽  
Pcr Product ◽  
Primer Sets ◽  
Species Specific

Abstract Two species of microsporidia, Nosema apis and Nosema ceranae, occur regularly and cause significant losses in apiculture throughout the world. N. ceranae is thought to be an emerging pathogen of the European honey bee which is replacing N. apis. Microscopic analysis of honey bees collected in Tyumen region, South-Western Siberia, suggested presence of two microsporidial pathogens slightly differing in spore size and shape. PCR detection using species-specific primer sets 312APIS and 218MITOC followed by PCR product sequencing confirmed the diagnosis of N. apis and N. ceranae, respectively. Microsporidia were present in private apiaries through 2008-2010, and among 21 colonies from 7 localities, two colonies were infected with both pathogens, while infections with N. apis only were detected in 8, and with N. ceranae only in 13 colonies. These data suggest that N. ceranae is widely spread in South-Western Siberia alongside with N. apis and is able to persist in the regions with average January temperatures below –18°C.

Design of specific primer sets for SARS-CoV-2 variants using evolutionary algorithms

2021 ◽  
Author(s):  
Alejandro Lopez Rincon ◽  
Carmina A. Perez Romero ◽  
Lucero Mendoza Maldonado ◽  
Eric Claassen ◽  
Johan Garssen ◽  
...  
Keyword(s):  
Evolutionary Algorithms ◽  
Specific Primer ◽  
Primer Sets

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus

Plant Disease ◽  
2007 ◽  
Vol 91 (7) ◽  
pp. 865-870 ◽  
Author(s):  
J. W. Hyun ◽  
N. A. Peres ◽  
S.-Y. Yi ◽  
L. W. Timmer ◽  
K. S. Kim ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Sweet Orange ◽  
Random Amplified Polymorphic Dna ◽  
Its Region ◽  
The United States ◽  
Specific Primer ◽  
Identification Of Species ◽  
Orange Fruit ◽  
Pcr Assays ◽  
Primer Sets

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River

2018 ◽  
Vol 15 (8) ◽  
pp. 1739 ◽  
Author(s):  
Hae-Kyung Park ◽  
Mi-Ae Kwon ◽  
Hae-Jin Lee ◽  
Jonghee Oh ◽  
Su-Heon Lee ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Water Samples ◽  
Its Region ◽  
Cyanobacterial Blooms ◽  
Specific Primer ◽  
Nakdong River ◽  
Off Flavors ◽  
Primer Sets ◽  
The Nakdong River ◽  
Harmful Cyanobacterial Blooms

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

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