High-Throughput Illumina MiSeq Amplicon Sequencing of Yeast Communities Associated With Indigenous Dairy Products From Republics of Benin and Niger

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.

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A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

10.1101/801175 ◽

2019 ◽

Author(s):

Abdul Wahab ◽

Ayaz Shaukat ◽

Qasim Ali ◽

Mubashir Hussain ◽

Taj Ali Khan ◽

...

Keyword(s):

Dna Sequencing ◽

High Throughput ◽

Disease Transmission ◽

Diagnostic Performance ◽

Detection Threshold ◽

Plasmodium Species ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Diagnostic Tools ◽

Sequencing Method

AbstractVarious PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput ‘haemoprotobiome’ metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

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Identifying Hidden Viable Bacterial Taxa in Tropical Forest Soils Using Amplicon Sequencing of Enrichment Cultures

Biology ◽

10.3390/biology10070569 ◽

2021 ◽

Vol 10 (7) ◽

pp. 569

Author(s):

Chakriya Sansupa ◽

Sara Fareed Mohamed Wahdan ◽

Terd Disayathanoowat ◽

Witoon Purahong

Keyword(s):

Bacterial Community ◽

Forest Soil ◽

Culture Media ◽

Enrichment Culture ◽

Sequence Similarity ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Soil Samples ◽

Enrichment Cultures ◽

Total Community

This study aims to estimate the proportion and diversity of soil bacteria derived from eDNA-based and culture-based methods. Specifically, we used Illumina Miseq to sequence and characterize the bacterial communities from (i) DNA extracted directly from forest soil and (ii) DNA extracted from a mixture of bacterial colonies obtained by enrichment cultures on agar plates of the same forest soil samples. The amplicon sequencing of enrichment cultures allowed us to rapidly screen a culturable community in an environmental sample. In comparison with an eDNA community (based on a 97% sequence similarity threshold), the fact that enrichment cultures could capture both rare and abundant bacterial taxa in forest soil samples was demonstrated. Enrichment culture and eDNA communities shared 2% of OTUs detected in total community, whereas 88% of enrichment cultures community (15% of total community) could not be detected by eDNA. The enrichment culture-based methods observed 17% of the bacteria in total community. FAPROTAX functional prediction showed that the rare and unique taxa, which were detected with the enrichment cultures, have potential to perform important functions in soil systems. We suggest that enrichment culture-based amplicon sequencing could be a beneficial approach to evaluate a cultured bacterial community. Combining this approach together with the eDNA method could provide more comprehensive information of a bacterial community. We expected that more unique cultured taxa could be detected if further studies used both selective and non-selective culture media to enrich bacteria at the first step.

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Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽

10.3390/fuels2020014 ◽

2021 ◽

Vol 2 (2) ◽

pp. 241-252

Author(s):

Dyah Asri Handayani Taroepratjeka ◽

Tsuyoshi Imai ◽

Prapaipid Chairattanamanokorn ◽

Alissara Reungsang

Keyword(s):

Microbial Communities ◽

High Throughput ◽

High Throughput Sequencing ◽

High Salinity ◽

Amplicon Sequencing ◽

Spatial Proximity ◽

Lignocellulosic Waste ◽

Evaporation Pond ◽

Operational Taxonomic Units ◽

Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

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DNA extraction from concrete v2

10.17504/protocols.io.b2i5qcg6 ◽

2021 ◽

Author(s):

Anders Kiledal ◽

Julia A Maresca

Keyword(s):

Dna Extraction ◽

Dental Plaque ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Metagenomic Sequencing ◽

Sample Analysis ◽

Larger Sample ◽

Laboratory Contamination ◽

Biomass Sample ◽

Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

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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’.

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Combination of Multiple Ligation-Dependent Probe Amplification and Illumina MiSeq Amplicon Sequencing for TSC1/TSC2 Gene Analyses in Patients with Tuberous Sclerosis Complex

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2016.10.009 ◽

2017 ◽

Vol 19 (2) ◽

pp. 265-276 ◽

Cited By ~ 5

Author(s):

Nur Farrah Dila Ismail ◽

Abdul Qawee Rani ◽

Nik Mohd Ariff Nik Abdul Malik ◽

Chia Boon Hock ◽

Siti Nabilahuda Mohd Azlan ◽

...

Keyword(s):

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Tsc2 Gene ◽

Dependent Probe

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Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

Applied and Environmental Microbiology ◽

10.1128/aem.00905-17 ◽

2017 ◽

Vol 83 (17) ◽

Cited By ~ 40

Author(s):

Francesca De Filippis ◽

Manolo Laiola ◽

Giuseppe Blaiotta ◽

Danilo Ercolini

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Biological Samples ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

Amplicon Sequencing ◽

Fungal Species ◽

Fungal Communities ◽

Its2 Region ◽

Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

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1621 Comparisons of microbial populations found in the rumen and in a dual-flow continuous culture fermentation system using high-throughput 16S amplicon sequencing

Journal of Animal Science ◽

10.2527/jam2016-1621 ◽

2016 ◽

Vol 94 (suppl_5) ◽

pp. 789-789

Author(s):

I. J. Salfer ◽

H. E. Larson ◽

M. D. Stern

Keyword(s):

Continuous Culture ◽

High Throughput ◽

Amplicon Sequencing ◽

Microbial Populations ◽

Fermentation System ◽

16S Amplicon Sequencing

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UNCROSS: Filtering of high-frequency cross-talk in 16S amplicon reads

10.1101/088666 ◽

2016 ◽

Cited By ~ 20

Author(s):

Robert C. Edgar

Keyword(s):

Ribosomal Rna ◽

Cross Talk ◽

High Frequency ◽

Biological Diversity ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Illumina Gaiix ◽

Microbial Metagenomics ◽

Tumor Sequencing ◽

Rna I

AbstractNext-generation amplicon sequencing is widely used for surveying biological diversity in applications such as microbial metagenomics, immune system repertoire analysis and targeted tumor sequencing of cancer-associated genes. In such studies, assignment of reads to incorrect samples (cross-talk) is a well-documented problem that is rarely considered in practice. By considering unexpected OTUs in artificial (mock) samples, I estimate that cross-talk occurred for ~2% of the reads in one Illumina GAIIx run and eleven Illumina MiSeq runs targeting 16S ribosomal RNA. I also describe UNCROSS, an algorithm for detecting and filtering cross-talk in OTU tables.

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