Faculty Opinions recommendation of Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform.

ABSTRACT Amplification, 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 those 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, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon 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 that 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 those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for 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.

Download Full-text

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

10.1101/417618 ◽

2018 ◽

Cited By ~ 3

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.

Download Full-text

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

PLoS ONE ◽

10.1371/journal.pone.0176716 ◽

2017 ◽

Vol 12 (4) ◽

pp. e0176716 ◽

Cited By ~ 50

Author(s):

Chongqing Wen ◽

Liyou Wu ◽

Yujia Qin ◽

Joy D. Van Nostrand ◽

Daliang Ning ◽

...

Keyword(s):

Illumina Miseq ◽

Amplicon Sequencing ◽

Illumina Miseq Platform ◽

Miseq Platform

Download Full-text

Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform

Nucleic Acids Research ◽

10.1093/nar/gku1341 ◽

2015 ◽

Vol 43 (6) ◽

pp. e37-e37 ◽

Cited By ~ 405

Author(s):

Melanie Schirmer ◽

Umer Z. Ijaz ◽

Rosalinda D'Amore ◽

Neil Hall ◽

William T. Sloan ◽

...

Keyword(s):

Illumina Miseq ◽

Amplicon Sequencing ◽

Sequencing Errors ◽

Illumina Miseq Platform ◽

Miseq Platform ◽

Insight Into

Download Full-text

A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0395 ◽

2018 ◽

Vol 374 (1763) ◽

pp. 20170395 ◽

Cited By ~ 9

Author(s):

Barnabas H. Daru ◽

Elizabeth A. Bowman ◽

Donald H. Pfister ◽

A. Elizabeth Arnold

Keyword(s):

Species Interactions ◽

Fungal Endophytes ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Herbarium Specimens ◽

Proof Of Concept ◽

Ledum Palustre ◽

Surface Contaminants ◽

Symptomless Plant ◽

Miseq Platform

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome ( Andromeda polifolia and Ledum palustre subsp . groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years . We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene. This article is part of the theme issue ‘Biological collections for understanding biodiversity in the Anthropocene’.

Download Full-text

Detection and monitoring of insect traces in bioaerosols

PeerJ ◽

10.7717/peerj.10862 ◽

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.

Download Full-text

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

Microorganisms ◽

10.3390/microorganisms8091363 ◽

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.

Download Full-text

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

Scientific Data ◽

10.1038/s41597-020-00695-9 ◽

2020 ◽

Vol 7 (1) ◽

Cited By ~ 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.

Download Full-text

A Plant Growth-Promoting Microbial Soil Amendment Dynamically Alters the Strawberry Root Bacterial Microbiome

Scientific Reports ◽

10.1038/s41598-019-53623-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Siwen Deng ◽

Heidi M.-L. Wipf ◽

Grady Pierroz ◽

Ted K. Raab ◽

Rajnish Khanna ◽

...

Keyword(s):

Bacterial Community ◽

Soil Amendment ◽

Bacterial Community Composition ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Microbiome ◽

Plant Growth Promoting ◽

Miseq Platform ◽

Soil Rhizosphere

AbstractDespite growing interest in utilizing microbial-based methods for improving crop growth, much work still remains in elucidating how beneficial plant-microbe associations are established, and what role soil amendments play in shaping these interactions. Here, we describe a set of experiments that test the effect of a commercially available soil amendment, VESTA, on the soil and strawberry (Fragaria x ananassa Monterey) root bacterial microbiome. The bacterial communities of the soil, rhizosphere, and root from amendment-treated and untreated fields were profiled at four time points across the strawberry growing season using 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. In all sample types, bacterial community composition and relative abundance were significantly altered with amendment application. Importantly, time point effects on composition are more pronounced in the root and rhizosphere, suggesting an interaction between plant development and treatment effect. Surprisingly, there was slight overlap between the taxa within the amendment and those enriched in plant and soil following treatment, suggesting that VESTA may act to rewire existing networks of organisms through an, as of yet, uncharacterized mechanism. These findings demonstrate that a commercial microbial soil amendment can impact the bacterial community structure of both roots and the surrounding environment.

Download Full-text