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

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

mSystems ◽

10.1128/msystems.00029-19 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 17

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.

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

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

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727561867.793570406 ◽

2020 ◽

Author(s):

Matthew Horton

Keyword(s):

Illumina Miseq ◽

Amplicon Sequencing ◽

Illumina Miseq Platform ◽

Miseq Platform

Download Full-text

A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

10.1101/142356 ◽

2017 ◽

Cited By ~ 1

Author(s):

Gregory L. Owens ◽

Marco Todesco ◽

Emily B. M. Drummond ◽

Sam Yeaman ◽

Loren H. Rieseberg

Keyword(s):

Allele Frequency ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequence Data ◽

Molecular Ecology ◽

Whole Genome Shotgun ◽

Whole Genome ◽

Illumina Hiseq ◽

Illumina Hiseq Platform ◽

Post Hoc

AbstractHigh throughput sequencing using the Illumina HiSeq platform is a pervasive and critical molecular ecology resource, and has provided the data underlying many recent advances. A recent study has suggested that ‘index switching’, where reads are misattributed to the wrong sample, may be higher in new versions of the HiSeq platform. This has the potential to invalidate both published and in-progress work across the field. Here, we test for evidence of index switching in an exemplar whole genome shotgun dataset sequenced on both the Illumina HiSeq 2500, which should not have the problem, and the Illumina HiSeq X, which may. We leverage unbalanced heterozygotes, which may be produced by index switching, and ask whether the under-sequenced allele is more likely to be found in other samples in the same lane than expected based on the allele frequency. Although we validate the sensitivity of this method using simulations, we find that neither the HiSeq 2500 nor the HiSeq X have evidence of index switching. This suggests that, thankfully, index switching may not be a ubiquitous problem in HiSeq X sequence data. Lastly, we provide scripts for applying our method so that index switching can be tested for in other datasets.

Download Full-text

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

10.1101/2020.11.13.370387 ◽

2020 ◽

Author(s):

Justin P. Shaffer ◽

Clarisse Marotz ◽

Pedro Belda-Ferre ◽

Cameron Martino ◽

Stephen Wandro ◽

...

Keyword(s):

Microbial Community ◽

High Throughput ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Sequence Data ◽

Rna Virus ◽

Microbial Community Composition ◽

Rna Extraction ◽

Acid Extraction ◽

Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

Download Full-text

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

Microbiome ◽

10.1186/2049-2618-2-6 ◽

2014 ◽

Vol 2 (1) ◽

pp. 6 ◽

Cited By ~ 671

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

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.01753-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6324-6332 ◽

Cited By ~ 49

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.

Download Full-text