scholarly journals A Comprehensive Evaluation of Single-end Sequencing Data Analyses for Environmental Microbiome Research

2021 ◽  
Author(s):  
Meganathan Ramakodi
Keyword(s):  
Sample Size ◽  
Comprehensive Evaluation ◽  
Illumina Miseq ◽  
Sequencing Data ◽  
Core Microbiome ◽  
Data Analyses ◽  
Illumina Miseq Platform ◽  
Microbiome Research ◽  
Miseq Platform ◽  
Sequencing Platforms

Abstract Illumina sequencing platforms have been widely used for amplicon-based environmental microbiome research. Analyses of amplicon data of environmental samples, generated from Illumina MiSeq platform illustrate the reverse (R2) reads in the PE datasets to have low quality towards the 3’ end of the reads which affect the sequencing depth of samples and ultimately impact the sample size which may possibly lead to an altered outcome. This study evaluates the usefulness of single-end (SE) sequencing data in microbiome research when the Illumina MiSeq PE dataset shows significantly high number of low quality reverse reads. In this study, the amplicon data (V1V3, V3V4, V4V5 and V6V8) from 128 environmental (soil) samples, downloaded from SRA, demonstrate the efficiency of single-end (SE) sequencing data analyses in microbiome research. The SE datasets were found to infer the core microbiome structure as comparable to the PE dataset. Conspicuously, the forward (R1) datasets inferred a higher number of taxa as compared to PE datasets for most of the amplicon regions, except V3V4. Thus, analyses of SE sequencing data, especially R1 reads, in environmental microbiome studies could ameliorate the problems arising on sample size of the study due to low quality reverse reads in the dataset. However, care must be taken while interpreting the microbiome structure as few taxa observed in the PE datasets were absent in the SE datasets. In conclusion, this study demonstrates the availability of choices in analyzing the amplicon data without having the need to remove samples with low quality reverse reads.

cpn60 metagenomic amplicon library preparation for the Illumina Miseq platform

2021 ◽  
Author(s):  
Champika Fernando ◽  
Janet E. Hill
Keyword(s):  
Dna Sequences ◽  
Illumina Miseq ◽  
Library Preparation ◽  
Illumina Miseq Platform ◽  
Wide Range ◽  
Miseq Platform ◽  
Identification Of Bacteria ◽  
Sequencing Platforms ◽  
Almost All ◽  
Level Identification

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.

scholarly journals Detection and monitoring of insect traces in bioaerosols

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10862
Author(s):  
Panyapon Pumkaeo ◽  
Junko Takahashi ◽  
Hitoshi Iwahashi
Keyword(s):  
Aerosol Particles ◽  
Illumina Miseq ◽  
Identification Accuracy ◽  
Insect Community ◽  
Cytochrome C Oxidase I ◽  
True Bugs ◽  
Illumina Miseq Platform ◽  
Miseq Platform ◽  
Pm2.5 And Pm10 ◽  
First Time

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.

scholarly journals Faecal Microbiota of Dogs Offered a Vegetarian Diet with or without the Supplementation of Feather Meal and either Cornmeal, Rye or Fermented Rye: A Preliminary Study

2020 ◽  
Vol 8 (9) ◽  
pp. 1363
Author(s):  
Julia Hankel ◽  
Amr Abd El-Wahab ◽  
Richard Grone ◽  
Birgit Keller ◽  
Eric Galvez ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Vegetarian Diet ◽  
Rrna Gene ◽  
Feather Meal ◽  
Illumina Miseq Platform ◽  
Faecal Samples ◽  
Miseq Platform ◽  
Preliminary Study

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.

scholarly journals Viral metagenomes of Lake Soyang, the largest freshwater lake in South Korea

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Kira Moon ◽  
Suhyun Kim ◽  
Ilnam Kang ◽  
Jang-Cheon Cho
Keyword(s):  
South Korea ◽  
Illumina Miseq ◽  
Freshwater Lakes ◽  
Genome Sequences ◽  
Illumina Miseq Platform ◽  
Viral Community ◽  
Viral Communities ◽  
Miseq Platform ◽  
Metagenome Sequencing ◽  
Freshwater Reservoir

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.

scholarly journals Response of Fecal Bacterial Flora to the Exposure of Fumonisin B1 in BALB/c Mice

Toxins ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 612
Author(s):  
Fan Zhang ◽  
Zhiwei Chen ◽  
Lin Jiang ◽  
Zihan Chen ◽  
Hua Sun
Keyword(s):  
Animal Health ◽  
Bacterial Flora ◽  
Fumonisin B1 ◽  
Illumina Miseq ◽  
Fecal Microbiota ◽  
Control Group ◽  
Illumina Miseq Platform ◽  
Regulatory Limits ◽  
Miseq Platform ◽  
High Diversity

Fumonisins are a kind of mycotoxin that has harmful influence on the health of humans and animals. Although some research studies associated with fumonisins have been reported, the regulatory limits of fumonisins are imperfect, and the effects of fumonisins on fecal bacterial flora of mice have not been suggested. In this study, in order to investigate the effects of fumonisin B1 (FB1) on fecal bacterial flora, BALB/c mice were randomly divided into seven groups, which were fed intragastrically with 0 mg/kg, 0.018 mg/kg, 0.054 mg/kg, 0.162 mg/kg, 0.486 mg/kg, 1.458 mg/kg and 4.374 mg/kg of FB1 solutions, once a day for 8 weeks. Subsequently, feces were collected for analysis of microflora. The V3-V4 16S rRNA of fecal bacterial flora was sequenced using the Illumina MiSeq platform. The results revealed that fecal bacterial flora of mice treated with FB1 presented high diversity. Additionally, the composition of fecal bacterial flora of FB1 exposure groups showed marked differences from that of the control group, especially for the genus types including Alloprevotella, Prevotellaceae_NK3B31_group, Rikenellaceae_RC9_gut_group, Parabacteroides and phylum types including Cyanobacteria. In conclusion, our data indicate that FB1 alters the diversity and composition of fecal microbiota in mice. Moreover, the minimum dose of FB1 exposure also causes changes in fecal microbiota to some extent. This study is the first to focus on the dose-related effect of FB1 exposure on fecal microbiota in rodent animals and gives references to the regulatory doses of fumonisins for better protection of human and animal health.

scholarly journals Baseline Data of the Fungal Phytobiome of Three Sorghum (Sorghum bicolor) Cultivars in South Africa Using Targeted Environmental Sequencing

2021 ◽  
Vol 7 (11) ◽  
pp. 978
Author(s):  
Gilmore T. Pambuka ◽  
Tonjock Rosemary Kinge ◽  
Soumya Ghosh ◽  
Errol D. Cason ◽  
Martin M. Nyaga ◽  
...  
Keyword(s):  
Crop Production ◽  
Plant Pathogens ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Targeted Sequencing ◽  
Illumina Miseq Platform ◽  
Below Ground ◽  
Miseq Platform ◽  
Low Levels ◽  
Plant Surfaces

Plant-associated fungi, or the mycobiome, inhabit plant surfaces above ground, reside in plant tissues as endophytes, or are rhizosphere in the narrow zone of soil surrounding plant roots. Studies have characterized mycobiomes of various plant species, but little is known about the sorghum mycobiome, especially in Africa, despite sorghum being one of the most important indigenous and commercial cereals in Africa. In this study, the mycobiome associated with above- and below-ground tissues of three commercial sorghum cultivars, as well as from rhizosphere and surrounding bulk soil samples, were sequenced using targeted sequencing with the Illumina MiSeq platform. Relative abundance differences between fungal communities were found between above-ground and below-ground niches, with most differences mostly in the dominant MOTUs, such as Davidiellaceae sp. (Cladosporium), Didymellaceae sp. 1 (Phoma), Fusarium, Cryptococcus and Mucor. Above-ground communities also appeared to be more diverse than below-ground communities, and plants harboured the most diversity. A considerable number of MOTUs were shared between the cultivars although, especially for NS5511, their abundances often differed. Several of the detected fungal groups include species that are plant pathogens of sorghum, such as Fusarium, and, at low levels, Alternaria and the Ustilaginomycetes. Findings from this study illustrate the usefulness of targeted sequencing of the ITS rDNA gene region (ITS2) to survey and monitor sorghum fungal communities and those from associated soils. This knowledge may provide tools for disease management and crop production and improvement.

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