A clinician's guide to microbiome analysis Claesson, Marcus J; Clooney, Adam G; O'Toole, Paul W
Nature reviews. Gastroenterology & hepatology,
10/2017, Volume:
14, Issue:
10
Journal Article
Peer reviewed
Microbiome analysis involves determining the composition and function of a community of microorganisms in a particular location. For the gastroenterologist, this technology opens up a rapidly ...evolving set of challenges and opportunities for generating novel insights into the health of patients on the basis of microbiota characterizations from intestinal, hepatic or extraintestinal samples. Alterations in gut microbiota composition correlate with intestinal and extraintestinal disease and, although only a few mechanisms are known, the microbiota are still an attractive target for developing biomarkers for disease detection and management as well as potential therapeutic applications. In this Review, we summarize the major decision points confronting new entrants to the field or for those designing new projects in microbiome research. We provide recommendations based on current technology options and our experience of sequencing platform choices. We also offer perspectives on future applications of microbiome research, which we hope convey the promise of this technology for clinical applications.
Alterations in the human intestinal microbiota are linked to conditions including inflammatory bowel disease, irritable bowel syndrome, and obesity. The microbiota also undergoes substantial changes ...at the extremes of life, in infants and older people, the ramifications of which are still being explored. We applied pyrosequencing of over 40,000 16S rRNA gene V4 region amplicons per subject to characterize the fecal microbiota in 161 subjects aged 65 y and older and 9 younger control subjects. The microbiota of each individual subject constituted a unique profile that was separable from all others. In 68% of the individuals, the microbiota was dominated by phylum Bacteroides, with an average proportion of 57% across all 161 baseline samples. Phylum Firmicutes had an average proportion of 40%. The proportions of some phyla and genera associated with disease or health also varied dramatically, including Proteobacteria, Actinobacteria, and FAECALIBACTERIA: The core microbiota of elderly subjects was distinct from that previously established for younger adults, with a greater proportion of Bacteroides spp. and distinct abundance patterns of Clostridium groups. Analyses of 26 fecal microbiota datasets from 3-month follow-up samples indicated that in 85% of the subjects, the microbiota composition was more like the corresponding time-0 sample than any other dataset. We conclude that the fecal microbiota of the elderly shows temporal stability over limited time in the majority of subjects but is characterized by unusual phylum proportions and extreme variability.
Shotgun metagenomic sequencing reveals the potential in microbial communities. However, lower-cost 16S ribosomal RNA (rRNA) gene sequencing provides taxonomic, not functional, observations. To remedy ...this, we previously introduced Piphillin, a software package that predicts functional metagenomic content based on the frequency of detected 16S rRNA gene sequences corresponding to genomes in regularly updated, functionally annotated genome databases. Piphillin (and similar tools) have previously been evaluated on 16S rRNA data processed by the clustering of sequences into operational taxonomic units (OTUs). New techniques such as amplicon sequence variant error correction are in increased use, but it is unknown if these techniques perform better in metagenomic content prediction pipelines, or if they should be treated the same as OTU data in respect to optimal pipeline parameters.
To evaluate the effect of 16S rRNA sequence analysis method (clustering sequences into OTUs vs amplicon sequence variant error correction into amplicon sequence variants (ASVs)) on the ability of Piphillin to predict functional metagenomic content, we evaluated Piphillin-predicted functional content from 16S rRNA sequence data processed through OTU clustering and error correction into ASVs compared to corresponding shotgun metagenomic data. We show a strong correlation between metagenomic data and Piphillin-predicted functional content resulting from both 16S rRNA sequence analysis methods. Differential abundance testing with Piphillin-predicted functional content exhibited a low false positive rate (< 0.05) while capturing a large fraction of the differentially abundant features resulting from corresponding metagenomic data. However, Piphillin prediction performance was optimal at different cutoff parameters depending on 16S rRNA sequence analysis method. Using data analyzed with amplicon sequence variant error correction, Piphillin outperformed comparable tools, for instance exhibiting 19% greater balanced accuracy and 54% greater precision compared to PICRUSt2.
Our results demonstrate that raw Illumina sequences should be processed for subsequent Piphillin analysis using amplicon sequence variant error correction (with DADA2 or similar methods) and run using a 99% ID cutoff for Piphillin, while sequences generated on platforms other than Illumina should be processed via OTU clustering (e.g., UPARSE) and run using a 96% ID cutoff for Piphillin. Piphillin is publicly available for academic users (Piphillin server. http://piphillin.secondgenome.com/.).
Stipitate kelp species such as Laminaria digitata dominate most cold-water subtidal rocky shores and form underwater forests which are among the most productive coastal systems worldwide. Laminaria ...also sustains rich bacterial communities which offer a variety of biotechnological applications. However, to date, in-depth studies on the diversity and uniqueness of bacterial communities associated with this macroalgal species, their ecological role and their interactions with the alga are under-represented. To address this, the epibacterial populations associated with different thallus regions (holdfast, stipe, meristem, blade) of this brown seaweed were investigated using high-throughput Illumina sequencing of the 16S rRNA genes. The results show that epibacterial communities of the brown seaweed are significantly different and specific to the thallus region, with the shared bacterial population comprising of only 1.1% of the total amplicon sequence variants. The diverse holdfast and blade tissues formed distinct clusters while the meristem and stipe regions are more closely related. The data obtained further supports the hypothesis that macroalgal bacterial communities are shaped by morphological niches and display specificity.
Taxonomic classification is a corner stone for the characterisation and comparison of microbial communities. Currently, most existing methods are either slow, restricted to specific communities, ...highly sensitive to taxonomic inconsistencies, or limited to genus level classification. As crucial microbiota information is hinging on high-level resolution it is imperative to increase taxonomic resolution to species level wherever possible.
In response to this need we developed SPINGO, a flexible and stand-alone software dedicated to high-resolution assignment of sequences to species level using partial 16S rRNA gene sequences from any environment. SPINGO compares favourably to other methods in terms of classification accuracy, and is as fast or faster than those that have higher error rates. As a demonstration of its flexibility for other types of target genes we successfully applied SPINGO also on cpn60 amplicon sequences.
SPINGO is an accurate, flexible and fast method for low-level taxonomic assignment. This combination is becoming increasingly important for rapid and accurate processing of amplicon data generated by newer next generation sequencing technologies.
The human gut microbiome is a complex ecosystem of microorganisms that play an important role in human health, influencing functions such as vitamin uptake, digestion and immunomodulation. While ...research of the gut microbiome has expanded considerably over the past decade, some areas such as the relationship between exercise and the microbiome remain relatively under investigated. Despite this, multiple studies have shown a potential bidirectional relationship between exercise and the gut microbiome, with some studies demonstrating the possibility of influencing this relationship. This, in turn, could provide a useful route to influence athletic performance via microbiome manipulation, a valuable prospect for many elite athletes and their teams. The evidence supporting the potential benefits of pursuing this route and associated future perspectives are discussed in this review.
The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used ...to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing
in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic
and
strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization.
Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products.
Highlights • The amygdala is an important brain region for emotional behaviour, such as social life and anxiety, and is activated by social interaction. • RNA-sequencing in the mouse amygdala ...revealed differential gene expression in germ-free animals. • Upregulated genes were associated with increased neuronal activity. • Differences in RNA editing were associated with neuronal structure and function. • Colonisation with a conventional microbiota after weaning fails to completely restore transcriptome alterations.
Our understanding of the composition and function of the human gut microbiota has improved dramatically in recent years. In this review, major milestones along this path will be reviewed. The ...methodological developments that underpinned this acceleration of scientific progress are discussed. We review the infant microbiota with special reference to colonization, succession and stabilization events. The healthy adult microbiota is then described, including reference to selected conditions that perturb the microbiota such as antibiotic treatment, bowel dysfunction and obesity. We put special emphasis on the special issues that characterize elderly subjects, including the challenges provided by “inflamm-aging”, with reference to how the microbiota might interact with these processes. Lastly, the elderly intestinal metagenomics project ELDERMET is described, which aims to establish the composition and function of the gut microbiota in several hundred Irish subjects.
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