Metagenomic Pyrosequencing and Microbial Identification

Abstract Background: The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies. Content: This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications. Summary: Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

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Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

Nova Biotechnologica et Chimica ◽

10.1515/nbec-2016-0003 ◽

2016 ◽

Vol 15 (1) ◽

pp. 23-34 ◽

Cited By ~ 7

Author(s):

Matej Planý ◽

Tomáš Kuchta ◽

Katarína Šoltýs ◽

Tomáš Szemes ◽

Domenico Pangallo ◽

...

Keyword(s):

16S Rrna ◽

16S Rdna ◽

Lactococcus Lactis ◽

Microbial Population ◽

Starter Culture ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Next Generation ◽

16S Rdna Amplicon Sequencing

Abstract Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in May) Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively). They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

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Coordination of microbe–host homeostasis by crosstalk with plant innate immunity

Nature Plants ◽

10.1038/s41477-021-00920-2 ◽

2021 ◽

Author(s):

Ka-Wai Ma ◽

Yulong Niu ◽

Yong Jia ◽

Jana Ordon ◽

Charles Copeland ◽

...

Keyword(s):

Growth Inhibition ◽

Amplicon Sequencing ◽

Host Susceptibility ◽

16S Rrna Genes ◽

Rrna Genes ◽

Root Growth Inhibition ◽

Natural Soil ◽

Molecular Pattern ◽

Immune Related Genes ◽

Molecular Patterns

AbstractPlants grown in natural soil are colonized by phylogenetically structured communities of microbes known as the microbiota. Individual microbes can activate microbe-associated molecular pattern (MAMP)-triggered immunity (MTI), which limits pathogen proliferation but curtails plant growth, a phenomenon known as the growth–defence trade-off. Here, we report that, in monoassociations, 41% (62 out of 151) of taxonomically diverse root bacterial commensals suppress Arabidopsis thaliana root growth inhibition (RGI) triggered by immune-stimulating MAMPs or damage-associated molecular patterns. Amplicon sequencing of bacterial 16S rRNA genes reveals that immune activation alters the profile of synthetic communities (SynComs) comprising RGI-non-suppressive strains, whereas the presence of RGI-suppressive strains attenuates this effect. Root colonization by SynComs with different complexities and RGI-suppressive activities alters the expression of 174 core host genes, with functions related to root development and nutrient transport. Furthermore, RGI-suppressive SynComs specifically downregulate a subset of immune-related genes. Precolonization of plants with RGI-suppressive SynComs, or mutation of one commensal-downregulated transcription factor, MYB15, renders the plants more susceptible to opportunistic Pseudomonas pathogens. Our results suggest that RGI-non-suppressive and RGI-suppressive root commensals modulate host susceptibility to pathogens by either eliciting or dampening MTI responses, respectively. This interplay buffers the plant immune system against pathogen perturbation and defence-associated growth inhibition, ultimately leading to commensal–host homeostasis.

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Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽

10.1186/s40168-021-01072-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Benjamin J. Callahan ◽

Dmitry Grinevich ◽

Siddhartha Thakur ◽

Michael A. Balamotis ◽

Tuval Ben Yehezkel

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Strain Identification ◽

Long Reads ◽

Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽

10.1186/s40168-020-00974-y ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Yusuke Okazaki ◽

Shohei Fujinaga ◽

Michaela M. Salcher ◽

Cristiana Callieri ◽

Atsushi Tanaka ◽

...

Keyword(s):

16S Rrna ◽

Regional Scale ◽

Scale Up ◽

Amplicon Sequencing ◽

Freshwater Ecosystems ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

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Impacts of switching tillage to no-tillage and vice versa on soil structure, enzyme activities, and prokaryotic community profiles in Argentinean semi-arid soils

FEMS Microbiology Ecology ◽

10.1093/femsec/fiab025 ◽

2021 ◽

Author(s):

L A Gabbarini ◽

E Figuerola ◽

J P Frene ◽

N B Robledo ◽

F M Ibarbalz ◽

...

Keyword(s):

Community Structure ◽

Soil Structure ◽

Management Practices ◽

Soil Health ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Prokaryotic Community ◽

C Cycle

Abstract The effects of tillage on soil structure, physiology, and microbiota structure were studied in a long-term field experiment, with side-to-side plots, established to compare effects of conventional tillage (CT) vs. no-till (NT) agriculture. After 27 years, part of the field under CT was switched to NT and vice versa. Soil texture, soil enzymatic profiles, and the prokaryotic community structure (16S rRNA genes amplicon sequencing) were analysed at two soil depths (0–5, 5–10 cm) in samples taken 6, 18, and 30 months after switching tillage practices. Soil enzymatic activities were higher in NT than CT, and enzymatic profiles responded to the changes much earlier than the overall prokaryotic community structure. Beta diversity measurements of the prokaryotic community indicated that the levels of stratification observed in long-term NT soils were already recovered in the new NT soils thirty months after switching from CT to NT. Bacteria and Archaea OTUs, which responded to NT were associated with coarse soil fraction, SOC and C cycle enzymes while CT responders were related to fine soil fractions and S cycle enzymes. This study showed the potential of managing the soil prokaryotic community and soil health through changes in agricultural management practices.

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16S rRNA gene sequences of Candidatus Methylumidiphilus (Methylococcales), a putative methanotrophic genus in lakes and ponds

10.1101/2021.09.28.462094 ◽

2021 ◽

Author(s):

Antti Juhani Rissanen ◽

Moritz Buck ◽

Sari Peura

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Stratified Lakes ◽

Link Type

A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds of boreal and subarctic area. However, it has probably escaped detection in many previous studies using 16S rRNA gene amplicon sequencing due to insufficient database coverage, which is because Ca. Methylumidiphilus lacks cultured representatives and previously analysed metagenome assembled genomes (MAGs) affiliated with it do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG dataset. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we could find 5 MAGs each containing one 1532 bp long sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in two unique 16S rRNA gene sequences. These sequences represented two different putative species, i.e. Ca. Methylumidiphilus alinenensis (Genbank accession: OK236221) as well as another so far unnamed species of Ca. Methylumidiphilus (Genbank accession: OK236220). We suggest that including these two sequences in reference databases will enhance 16S rRNA gene - based detection of members of this genus from environmental samples.

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Alpha- and Gammaproteobacterial Methanotrophs Codominate the Active Methane-Oxidizing Communities in an Acidic Boreal Peat Bog

Applied and Environmental Microbiology ◽

10.1128/aem.03640-15 ◽

2016 ◽

Vol 82 (8) ◽

pp. 2363-2371 ◽

Cited By ~ 37

Author(s):

Kaitlin C. Esson ◽

Xueju Lin ◽

Deepak Kumaresan ◽

Jeffrey P. Chanton ◽

J. Colin Murrell ◽

...

Keyword(s):

Dry Weight ◽

Amplicon Sequencing ◽

Stable Isotope Probing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Type I ◽

Type Ii ◽

Content Type ◽

Methane Consumption ◽

Field Samples

ABSTRACTThe objective of this study was to characterize metabolically active, aerobic methanotrophs in an ombrotrophic peatland in the Marcell Experimental Forest, in Minnesota. Methanotrophs were investigated in the field and in laboratory incubations using DNA-stable isotope probing (SIP), expression studies on particulate methane monooxygenase (pmoA) genes, and amplicon sequencing of 16S rRNA genes. Potential rates of oxidation ranged from 14 to 17 μmol of CH4g dry weight soil−1day−1. Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4%in situto 25 to 36% after 8 to 14 days. Phylogenetic analysis of the13C-enriched DNA fractions revealed that the active methanotrophs were dominated by the generaMethylocystis(type II;Alphaproteobacteria),Methylomonas, andMethylovulum(both, type I;Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed forpmoAin surface peat layers, which attenuated rapidly with depth, indicating that the highest methane consumption was associated with a depth of 0 to 10 cm. Metagenomes and sequencing of cDNApmoAamplicons from field samples confirmed that the dominant active methanotrophs wereMethylocystisandMethylomonas. Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the generaMethylomonasandMethylovulum(type I) can significantly contribute to aerobic methane oxidation in these ecosystems.

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Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes

Applied and Environmental Microbiology ◽

10.1128/aem.02953-09 ◽

2010 ◽

Vol 76 (12) ◽

pp. 3886-3897 ◽

Cited By ~ 165

Author(s):

Anna Y. Pei ◽

William E. Oberdorf ◽

Carlos W. Nossa ◽

Ankush Agarwal ◽

Pooja Chokshi ◽

...

Keyword(s):

16S Rrna ◽

Bacterial Species ◽

Human Microbiome ◽

Operational Definition ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Thermoanaerobacter Tengcongensis ◽

A Genome ◽

Prokaryotic Genomes

ABSTRACT Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp ). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

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Impact of Helicobacter Pylori Infection on Duodenal Microbial Community Structure and Microbial Metabolic Pathways

10.21203/rs.3.rs-166718/v2 ◽

2021 ◽

Author(s):

Tadashi Maeda ◽

Hiroaki Zai ◽

Yuto Fukui ◽

Yoshifumi Kato ◽

Eri Kumade ◽

...

Keyword(s):

Helicobacter Pylori ◽

16S Rrna ◽

Metabolic Pathways ◽

Amplicon Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Operational Taxonomic Units ◽

H Pylori ◽

The Impact

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection increased the relative mean abundance of Proteobacteria and Neisseria and decreased the abundance of the two other phyla (Actinobacteria and TM7) and nine genera (Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

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Phylotype-Level Characterization of Complex Lactobacilli Communities Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

10.1101/2020.09.09.290726 ◽

2020 ◽

Author(s):

Shaktheeshwari Silvaraju ◽

Nandita Menon ◽

Huan Fan ◽

Kevin Lim ◽

Sandra Kittelmann

Keyword(s):

High Resolution ◽

Amplicon Sequencing ◽

Trna Synthetase ◽

Species Level ◽

16S Rrna Genes ◽

Rrna Genes ◽

Low Resolution ◽

Shotgun Metagenomics ◽

Wide Range

ABSTRACTThe ‘lactobacilli’ to date encompass more than 270 closely related species that were recently re-classified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related, yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA-based amplicon sequencing have significant limitations (high cost, low resolution, etc.). Here, we generated a lactobacilli phylogeny based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of lactobacilli community diversity and structure at the species-level. This framework is based on a total of 445 pheS gene sequences, including sequences of 277 validly described species and subspecies (out of a total of 283, coverage of 98%). It allows differentiation between 263 lactobacilli species-level clades out of a total of 273 validly described species (including the proposed species L. timonensis) and a further two subspecies. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of ∼30,000 sequences, the minimum level of detection was approximately 0.02 pg per μl DNA (equalling approximately 10 genome copies per µl template DNA). The pheS approach along with parallel sequencing of partial 16S rRNA genes revealed a considerable lactobacilli diversity and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike.IMPORTANCESpecies within the former genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate for their exceptional functional diversity, the over 270 species were recently re-classified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost or copy number variations. The approach described here makes use of a single copy marker gene which outperforms other markers with regards to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address lactobacilli diversity and community structure in various environmental matrices. Such analyses can now be performed at broader scale to assess and monitor lactobacilli community assembly, structure and function at the species (in some cases even at sub-species) level across a wide range of academic and commercial applications.

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