scholarly journals Library preparation protocol to sequence V3-V4 region of 16S rRNA to run in Illumina MiSeq platform v1 (protocols.io.6i7hchn)

protocols.io ◽  
2019 ◽  
Author(s):  
Somasundhari Shanmuganandam ◽  
Benjamin Schwessinger ◽  
Robyn Hall
Author(s):  
Champika Fernando ◽  
Janet E. Hill

Abstract This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identification of bacteria. The primer cocktail used in this protocol amplifies cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identification. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quantification, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Specific instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.


Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 6 ◽  
Author(s):  
Douglas W Fadrosh ◽  
Bing Ma ◽  
Pawel Gajer ◽  
Naomi Sengamalay ◽  
Sandra Ott ◽  
...  

2017 ◽  
Vol 7 (2) ◽  
pp. 100-129 ◽  
Author(s):  
Alexis Rapin ◽  
Céline Pattaroni ◽  
Benjamin J. Marsland ◽  
Nicola L. Harris

2015 ◽  
Vol 53 (1) ◽  
pp. 60-69 ◽  
Author(s):  
Yoon-Seong Jeon ◽  
Sang-Cheol Park ◽  
Jeongmin Lim ◽  
Jongsik Chun ◽  
Bong-Soo Kim

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10862
Author(s):  
Panyapon Pumkaeo ◽  
Junko Takahashi ◽  
Hitoshi Iwahashi

Studies on bioaerosols have primarily focused on their chemical and biological compositions and their impact on public health and the ecosystem. However, most bioaerosol studies have only focused on viruses, bacteria, fungi, and pollen. To assess the diversity and composition of airborne insect material in particulate matter (PM) for the first time, we attempted to detect DNA traces of insect origin in dust samples collected over a two-year period. These samples were systematically collected at one-month intervals and categorized into two groups, PM2.5 and PM10, based on the aerodynamic diameter of the aerosol particles. Cytochrome-c oxidase I (COI) was the barcoding region used to identify the origins of the extracted DNA. The airborne insect community in these samples was analyzed using the Illumina MiSeq platform. The most abundant insect sequences belonged to the order Hemiptera (true bugs), whereas order Diptera were also detected in both PM2.5 and PM10 samples. Additionally, we inferred the presence of particulates of insect origin, such as brochosomes and integument particles, using scanning electron microscopy (SEM). This provided additional confirmation of the molecular results. In this study, we demonstrated the benefits of detection and monitoring of insect information in bioaerosols for understanding the source and composition. Our results suggest that the PM2.5 and PM10 groups are rich in insect diversity. Lastly, the development of databases can improve the identification accuracy of the analytical results.


2020 ◽  
Vol 8 (9) ◽  
pp. 1363
Author(s):  
Julia Hankel ◽  
Amr Abd El-Wahab ◽  
Richard Grone ◽  
Birgit Keller ◽  
Eric Galvez ◽  
...  

Anthropomorphism of dogs has affected feeding and the choice of components present in diets for dogs. Conflicting trends are present: raw or vegetarian appear more prevalent. Animal-derived proteins seem to have unfavourable impacts on intestinal microflora by decreasing the presence of Bacteroidetes. This preliminary study evaluates whether effects of diets with animal proteins on intestinal microbiota can be compensated by the addition of certain carbohydrates to dog diet. Eight female beagles were included in a cross-over study and fed a vegetarian diet or the same diet supplemented with feather meal (2.7%) and either 20% of cornmeal, fermented or non-fermented rye (moisture content of the diets about 42%). A 16S rRNA gene amplification was performed within the hypervariable region V4 on faecal samples and sequenced with the Illumina MiSeq platform. The Firmicutes/Bacteroidetes ratio tended to shift to the advantage of Firmicutes when feather meal and cornmeal were added (Firmicutes/Bacteroidetes ratio of 5.12 compared to 2.47 when offered the vegetarian diet) and tended to switch back to the advantage of Bacteroidetes if rye: fermented (2.17) or not (1.03) was added. The addition of rye might have the potential to compensate possible unfavourable effects of diets with animal proteins on intestinal microbiota of dogs.


2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Kira Moon ◽  
Suhyun Kim ◽  
Ilnam Kang ◽  
Jang-Cheon Cho

Abstract A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.


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