scholarly journals Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
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.

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

2019 ◽  
Vol 9 (1) ◽  
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.

scholarly journals An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 6 ◽  
Author(s):  
Douglas W Fadrosh ◽  
Bing Ma ◽  
Pawel Gajer ◽  
Naomi Sengamalay ◽  
Sandra Ott ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Illumina Miseq Platform ◽  
Miseq Platform ◽  
Rrna Gene Sequencing

scholarly journals Uterine Microbiota Progression from Calving until Establishment of Metritis in Dairy Cows

2015 ◽  
Vol 81 (18) ◽  
pp. 6324-6332 ◽  
Author(s):  
Soo Jin Jeon ◽  
Achilles Vieira-Neto ◽  
Mohanathas Gobikrushanth ◽  
Rodolfo Daetz ◽  
Rodolfo D. Mingoti ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Heat Map ◽  
Content Type ◽  
Illumina Miseq Platform ◽  
Uterine Health ◽  
Miseq Platform ◽  
The 16S Rrna Gene

ABSTRACTThe objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n= 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance ofProteobacteriaand an increase in the abundance ofBacteroidetesandFusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp.Bacteroideswas the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance ofBacteroidesorganisms increased, the uterine discharge score, a measure of uterine health, worsened.Fusobacteriumwas also an important genus associated with metritis becauseFusobacteriumabundance increased asBacteroidesabundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation withBacteroidesorFusobacteriumshowed that other bacteria, such asHelcoccocus,Filifactor, andPorphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as “CandidatusBlochmannia,”Escherichia,Sneathia, andPedobacter.

scholarly journals Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0176716 ◽  
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

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

2015 ◽  
Vol 43 (6) ◽  
pp. e37-e37 ◽  
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

scholarly journals PSIX-6 Gastrointestinal microbiome of calves fed solid diets containing different levels and sources of NDF

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 418-419
Author(s):  
Gercino F Virgínio Júnior ◽  
Milaine Poczynek ◽  
Ana Paula Silva ◽  
Ariany Toledo ◽  
Amanda Cezar ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Diversity Indices ◽  
Dairy Calves ◽  
Rrna Gene ◽  
Gastrointestinal Microbiome ◽  
Fecal Microbiome ◽  
Miseq Platform ◽  
Holstein Calves ◽  
Ad Libitum ◽  
Different Levels

Abstract Different levels and sources of NDF can modify the gastrointestinal microbiome. This study evaluated 18 Holstein calves housed in not-bedded suspended individual cages and fed one of three treatments: 22NDF - conventional starter containing 22% NDF (n = 7); 31NDF - starter with 31% NDF, replacing part of the corn by soybean hull (n = 6); and 22NDF+H - conventional starter with 22% NDF plus coast-cross hay ad libitum (n = 5). All animals received 4 L of milk replacer daily (24% CP; 18.5% fat; diluted to 12.5% solids), divided into two meals, being weaned at 8th week of age. After weaning, animals were housed in tropical shelters, fed with the respective solid diet and coast-cross hay ad libitum for all treatments. To evaluate the microbiome, ruminal fluid samples were collected using a modified Geishauser oral probe at weeks 2, 4, 6, 8 and 10, two hours after the morning feeding, and fecal samples were collected at birth (0) and at weeks 1, 2, 4, 8 and 10. The microbial community was determined by sequencing V3 and V4 region amplicons of the 16S rRNA gene that was amplified by PCR and sequenced by the Illumina MiSeq platform. Ruminal microbiome had no differences in diversity for the effects of weeks, treatments or interaction of both factors (Table 1). In feces, the diversity indices and evenness were higher for 22NDF+H when compared to 22NDF, with no difference for 31NDF. All indices were significantly affected by calves age. At birth, calves had the greatest diversity and richness. Week 1 and 2 had less evenness and diversity. Bacteroidota, Firmicutes_A and Firmicutes_C were the most abundant phylum in rumen and feces. The supply of hay was only effective in modifying the fecal microbiome of dairy calves, suggesting a resilience in the ruminal microbiome.

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

Sign in / Sign up

Export Citation Format

Share Document