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Open AccessArticle

Assessing the Accuracy of Quantitative Molecular Microbial Profiling

by Denise M. O'Sullivan, Thomas Laver, Sasithon Temisak, Nicholas Redshaw, Kathryn A. Harris, Carole A. Foy, David J. Studholme and Jim F. Huggett

Int. J. Mol. Sci. 2014, 15(11), 21476-21491; https://doi.org/10.3390/ijms151121476 - 21 Nov 2014

Cited by 15 | Viewed by 10011

Abstract

The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole [...] Read more.

The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessArticle

Metaphylogenomic and Potential Functionality of the Limpet Patella pellucida’s Gastrointestinal Tract Microbiome

by Magda Dudek, Jessica Adams, Martin Swain, Matthew Hegarty, Sharon Huws and Joe Gallagher

Int. J. Mol. Sci. 2014, 15(10), 18819-18839; https://doi.org/10.3390/ijms151018819 - 20 Oct 2014

Cited by 14 | Viewed by 8359

Abstract

This study investigated the microbial diversity associated with the digestive tract of the seaweed grazing marine limpet Patella pellucida. Using a modified indirect DNA extraction protocol and performing metagenomic profiling based on specific prokaryotic marker genes, the abundance of bacterial groups was [...] Read more.

This study investigated the microbial diversity associated with the digestive tract of the seaweed grazing marine limpet Patella pellucida. Using a modified indirect DNA extraction protocol and performing metagenomic profiling based on specific prokaryotic marker genes, the abundance of bacterial groups was identified from the analyzed metagenome. The members of three significantly abundant phyla of Proteobacteria, Firmicutes and Bacteroidetes were characterized through the literature and their predicted functions towards the host, as well as potential applications in the industrial environment assessed. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessArticle

Identification of Diversity-Generating Retroelements in Human Microbiomes

by Yuzhen Ye

Int. J. Mol. Sci. 2014, 15(8), 14234-14246; https://doi.org/10.3390/ijms150814234 - 15 Aug 2014

Cited by 27 | Viewed by 7568

Abstract

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by accelerating the evolution of target genes through a specialized, error-prone, reverse transcription process. First identified in a Bordetella phage (BPP-1), which mediates the phage tropism specificity [...] Read more.

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by accelerating the evolution of target genes through a specialized, error-prone, reverse transcription process. First identified in a Bordetella phage (BPP-1), which mediates the phage tropism specificity by generating variability in an involved gene, DGRs were predicted to be present in a larger collection of viral and bacterial species. A minimal DGR system is comprised of a reverse transcriptase (RTase) gene, a template sequence (TR) and a variable region (VR) within a target gene. We developed a computational tool, DGRscan, to allow either de novo identification (based on the prediction of potential template-variable region pairs) or similarity-based searches of DGR systems using known template sequences as the reference. The application of DGRscan to the human microbiome project (HMP) datasets resulted in the identification of 271 non-redundant DGR systems, doubling the size of the collection of known DGR systems. We further identified a large number of putative target genes (651, which share no more than 90% sequence identity at the amino acid level) that are potentially under diversification by the DGR systems. Our study provides the first survey of the DGR systems in the human microbiome, showing that the DGR systems are frequently found in human-associated bacterial communities, although they are of low incidence in individual genomes. Our study also provides functional clues for a large number of genes (reverse transcriptases and target genes) that were previously annotated as proteins of unknown functions or nonspecific functions. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessArticle

Exploring Neighborhoods in the Metagenome Universe

by Kathrin P. Aßhauer, Heiner Klingenberg, Thomas Lingner and Peter Meinicke

Int. J. Mol. Sci. 2014, 15(7), 12364-12378; https://doi.org/10.3390/ijms150712364 - 14 Jul 2014

Cited by 2 | Viewed by 8144

Abstract

The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of [...] Read more.

The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of the available sequence data alone. We have studied efficient sequence-based methods for large-scale identification of similar metagenomes within a database retrieval context. In a broad comparison of different profiling methods we found that vector-based distance measures are well-suitable for the detection of metagenomic neighbors. Our evaluation on more than 1700 publicly available metagenomes indicates that for a query metagenome from a particular habitat on average nine out of ten nearest neighbors represent the same habitat category independent of the utilized profiling method or distance measure. While for well-defined labels a neighborhood accuracy of 100% can be achieved, in general the neighbor detection is severely affected by a natural overlap of manually annotated categories. In addition, we present results of a novel visualization method that is able to reflect the similarity of metagenomes in a 2D scatter plot. The visualization method shows a similarly high accuracy in the reduced space as compared with the high-dimensional profile space. Our study suggests that for inspection of metagenome neighborhoods the profiling methods and distance measures can be chosen to provide a convenient interpretation of results in terms of the underlying features. Furthermore, supplementary metadata of metagenome samples in the future needs to comply with readily available ontologies for fine-grained and standardized annotation. To make profile-based k-nearest-neighbor search and the 2D-visualization of the metagenome universe available to the research community, we included the proposed methods in our CoMet-Universe server for comparative metagenome analysis. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessArticle

Generation and Analysis of Expressed Sequence Tags (ESTs) from Halophyte Atriplex canescens to Explore Salt-Responsive Related Genes

by Jingtao Li, Xinhua Sun, Gang Yu, Chengguo Jia, Jinliang Liu and Hongyu Pan

Int. J. Mol. Sci. 2014, 15(6), 11172-11189; https://doi.org/10.3390/ijms150611172 - 23 Jun 2014

Cited by 15 | Viewed by 6071

Abstract

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 [...] Read more.

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessArticle

A Comprehensive Insight into Tetracycline Resistant Bacteria and Antibiotic Resistance Genes in Activated Sludge Using Next-Generation Sequencing

by Kailong Huang, Junying Tang, Xu-Xiang Zhang, Ke Xu and Hongqiang Ren

Int. J. Mol. Sci. 2014, 15(6), 10083-10100; https://doi.org/10.3390/ijms150610083 - 05 Jun 2014

Cited by 61 | Viewed by 10083

Abstract

In order to comprehensively investigate tetracycline resistance in activated sludge of sewage treatment plants, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential tetracycline resistant bacteria (TRB) and antibiotic resistance genes (ARGs) in sludge cultured with different concentrations of tetracycline. Pyrosequencing [...] Read more.

In order to comprehensively investigate tetracycline resistance in activated sludge of sewage treatment plants, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential tetracycline resistant bacteria (TRB) and antibiotic resistance genes (ARGs) in sludge cultured with different concentrations of tetracycline. Pyrosequencing of 16S rRNA gene revealed that tetracycline treatment greatly affected the bacterial community structure of the sludge. Nine genera consisting of Sulfuritalea, Armatimonas, Prosthecobacter, Hyphomicrobium, Azonexus, Longilinea, Paracoccus, Novosphingobium and Rhodobacter were identified as potential TRB in the sludge. Results of qPCR, molecular cloning and metagenomic analysis consistently indicated that tetracycline treatment could increase both the abundance and diversity of the tet genes, but decreased the occurrence and diversity of non-tetracycline ARG, especially sulfonamide resistance gene sul2. Cluster analysis showed that tetracycline treatment at subinhibitory concentrations (5 mg/L) was found to pose greater effects on the bacterial community composition, which may be responsible for the variations of the ARGs abundance. This study indicated that joint use of 454 pyrosequencing and Illumina high-throughput sequencing can be effectively used to explore ARB and ARGs in the environment, and future studies should include an in-depth investigation of the relationship between microbial community, ARGs and antibiotics in sewage treatment plant (STP) sludge. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Review

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Open AccessReview

Viral Metagenomics on Animals as a Tool for the Detection of Zoonoses Prior to Human Infection?

by Sarah Temmam, Bernard Davoust, Jean-Michel Berenger, Didier Raoult and Christelle Desnues

Int. J. Mol. Sci. 2014, 15(6), 10377-10397; https://doi.org/10.3390/ijms150610377 - 10 Jun 2014

Cited by 50 | Viewed by 10230

Abstract

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of [...] Read more.

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of a bloodsucking arthropod. If a virus is able to adapt and replicate in its new human host, human-to-human transmissions may occur, possibly resulting in an epidemic, such as the A/H1N1 flu pandemic in 2009. Thus, predicting emerging zoonotic infections is an important challenge for public health officials in the coming decades. The recent development of viral metagenomics, i.e., the characterization of the complete viral diversity isolated from an organism or an environment using high-throughput sequencing technologies, is promising for the surveillance of such diseases and can be accomplished by analyzing the viromes of selected animals and arthropods that are closely in contact with humans. In this review, we summarize our current knowledge of viral diversity within such animals (in particular blood-feeding arthropods, wildlife and domestic animals) using metagenomics and present its possible future application for the surveillance of zoonotic and arboviral diseases. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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Open AccessReview

Marine Microbial Metagenomics: From Individual to the Environment

by Ching-Hung Tseng and Sen-Lin Tang

Int. J. Mol. Sci. 2014, 15(5), 8878-8892; https://doi.org/10.3390/ijms15058878 - 19 May 2014

Cited by 30 | Viewed by 8556

Abstract

Microbes are the most abundant biological entities on earth, therefore, studying them is important for understanding their roles in global ecology. The science of metagenomics is a relatively young field of research that has enjoyed significant effort since its inception in 1998. Studies [...] Read more.

Microbes are the most abundant biological entities on earth, therefore, studying them is important for understanding their roles in global ecology. The science of metagenomics is a relatively young field of research that has enjoyed significant effort since its inception in 1998. Studies using next-generation sequencing techniques on single genomes and collections of genomes have not only led to novel insights into microbial genomics, but also revealed a close association between environmental niches and genome evolution. Herein, we review studies investigating microbial genomics (largely in the marine ecosystem) at the individual and community levels to summarize our current understanding of microbial ecology in the environment. Full article

(This article belongs to the Special Issue Metagenomics: a Powerful Lens Viewing the Microbial World)

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