Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10[superscript 6] reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.National Institutes of Health (U.S.) (1R01HG005975-01)National Science Foundation (U.S.) (award #0743432)National Institutes of Health (U.S.) (grant NIHU54HG004969

Composition of the Adult Digestive Tract Bacterial Microbiome Based on Seven Mouth Surfaces, Tonsils, Throat and Stool Samples

Background: To understand the relationship between our bacterial microbiome and health, it is essential to define the microbiome in the absence of disease. The digestive tract includes diverse habitats and hosts the human body's greatest bacterial density. We describe the bacterial community composition of ten digestive tract sites from more than 200 normal adults enrolled in the Human Microbiome Project, and metagenomically determined metabolic potentials of four representative sites. Results: The microbiota of these diverse habitats formed four groups based on similar community compositions: buccal mucosa, keratinized gingiva, hard palate; saliva, tongue, tonsils, throat; sub- and supra-gingival plaques; and stool. Phyla initially identified from environmental samples were detected throughout this population, primarily TM7, SR1, and Synergistetes. Genera with pathogenic members were well-represented among this disease-free cohort. Tooth-associated communities were distinct, but not entirely dissimilar, from other oral surfaces. The Porphyromonadaceae, Veillonellaceae and Lachnospiraceae families were common to all sites, but the distributions of their genera varied significantly. Most metabolic processes were distributed widely throughout the digestive tract microbiota, with variations in metagenomic abundance between body habitats. These included shifts in sugar transporter types between the supragingival plaque, other oral surfaces, and stool; hydrogen and hydrogen sulfide production were also differentially distributed. Conclusions: The microbiomes of ten digestive tract sites separated into four types based on composition. A core set of metabolic pathways was present across these diverse digestive tract habitats. These data provide a critical baseline for future studies investigating local and systemic diseases affecting human health

Host genetic variation impacts microbiome composition across human body sites

Background: The composition of bacteria in and on the human body varies widely across human individuals, and has been associated with multiple health conditions. While microbial communities are influenced by environmental factors, some degree of genetic influence of the host on the microbiome is also expected. This study is part of an expanding effort to comprehensively profile the interactions between human genetic variation and the composition of this microbial ecosystem on a genome- and microbiome-wide scale. Results: Here, we jointly analyze the composition of the human microbiome and host genetic variation. By mining the shotgun metagenomic data from the Human Microbiome Project for host DNA reads, we gathered information on host genetic variation for 93 individuals for whom bacterial abundance data are also available. Using this dataset, we identify significant associations between host genetic variation and microbiome composition in 10 of the 15 body sites tested. These associations are driven by host genetic variation in immunity-related pathways, and are especially enriched in host genes that have been previously associated with microbiome-related complex diseases, such as inflammatory bowel disease and obesity-related disorders. Lastly, we show that host genomic regions associated with the microbiome have high levels of genetic differentiation among human populations, possibly indicating host genomic adaptation to environment-specific microbiomes. Conclusions: Our results highlight the role of host genetic variation in shaping the composition of the human microbiome, and provide a starting point toward understanding the complex interaction between human genetics and the microbiome in the context of human evolution and disease.</p

What traits are carried on mobile genetic elements, and why?

Although similar to any other organism, prokaryotes can transfer genes vertically from mother cell to daughter cell, they can also exchange certain genes horizontally. Genes can move within and between genomes at fast rates because of mobile genetic elements (MGEs). Although mobile elements are fundamentally self-interested entities, and thus replicate for their own gain, they frequently carry genes beneficial for their hosts and/or the neighbours of their hosts. Many genes that are carried by mobile elements code for traits that are expressed outside of the cell. Such traits are involved in bacterial sociality, such as the production of public goods, which benefit a cell's neighbours, or the production of bacteriocins, which harm a cell's neighbours. In this study we review the patterns that are emerging in the types of genes carried by mobile elements, and discuss the evolutionary and ecological conditions under which mobile elements evolve to carry their peculiar mix of parasitic, beneficial and cooperative genes

Current understanding of the human microbiome

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Medicine 24 (2018): 392–400, doi:10.1038/nm.4517.Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes, and mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this Review, we focus on studies in humans to describe these challenges, and propose strategies that leverage existing knowledge to move rapidly from correlation to causation, and ultimately to translation.Many of the studies described here in our laboratories were supported by the NIH, NSF, DOE, and the Alfred P. Sloan Foundation.2018-10-1

Complex host genetics influence the microbiome in inflammatory bowel disease

Background: Human genetics and host-associated microbial communities have been associated independently with a wide range of chronic diseases. One of the strongest associations in each case is inflammatory bowel disease (IBD), but disease risk cannot be explained fully by either factor individually. Recent findings point to interactions between host genetics and microbial exposures as important contributors to disease risk in IBD. These include evidence of the partial heritability of the gut microbiota and the conferral of gut mucosal inflammation by microbiome transplant even when the dysbiosis was initially genetically derived. Although there have been several tests for association of individual genetic loci with bacterial taxa, there has been no direct comparison of complex genome-microbiome associations in large cohorts of patients with an immunity-related disease. Methods: We obtained 16S ribosomal RNA (rRNA) gene sequences from intestinal biopsies as well as host genotype via Immunochip in three independent cohorts totaling 474 individuals. We tested for correlation between relative abundance of bacterial taxa and number of minor alleles at known IBD risk loci, including fine mapping of multiple risk alleles in the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene exon. We identified host polymorphisms whose associations with bacterial taxa were conserved across two or more cohorts, and we tested related genes for enrichment of host functional pathways. Results: We identified and confirmed in two cohorts a significant association between NOD2 risk allele count and increased relative abundance of Enterobacteriaceae, with directionality of the effect conserved in the third cohort. Forty-eight additional IBD-related SNPs have directionality of their associations with bacterial taxa significantly conserved across two or three cohorts, implicating genes enriched for regulation of innate immune response, the JAK-STAT cascade, and other immunity-related pathways. Conclusions: These results suggest complex interactions between genetically altered host functional pathways and the structure of the microbiome. Our findings demonstrate the ability to uncover novel associations from paired genome-microbiome data, and they suggest a complex link between host genetics and microbial dysbiosis in subjects with IBD across independent cohorts. Electronic supplementary material The online version of this article (doi:10.1186/s13073-014-0107-1) contains supplementary material, which is available to authorized users

Counting on the mental number line to make a move: sensorimotor ('pen') control and numerical processing

Mathematics is often conducted with a writing implement. But is there a relationship between numerical processing and sensorimotor ‘pen’ control? We asked participants to move a stylus so it crossed an unmarked line at a location specified by a symbolic number (1–9), where number colour indicated whether the line ran left–right (‘normal’) or vice versa (‘reversed’). The task could be simplified through the use of a ‘mental number line’ (MNL). Many modern societies use number lines in mathematical education and the brain’s representation of number appears to follow a culturally determined spatial organisation (so better task performance is associated with this culturally normal orientation—the MNL effect). Participants (counter-balanced) completed two consistent blocks of trials, ‘normal’ and ‘reversed’, followed by a mixed block where line direction varied randomly. Experiment 1 established that the MNL effect was robust, and showed that the cognitive load associated with reversing the MNL not only affected response selection but also the actual movement execution (indexed by duration) within the mixed trials. Experiment 2 showed that an individual’s motor abilities predicted performance in the difficult (mixed) condition but not the easier blocks. These results suggest that numerical processing is not isolated from motor capabilities—a finding with applied consequences

Low is large: spatial location and pitch interact in voice-based body size estimation

The binding of incongruent cues poses a challenge for multimodal perception. Indeed, although taller objects emit sounds from higher elevations, low-pitched sounds are perceptually mapped both to large size and to low elevation. In the present study, we examined how these incongruent vertical spatial cues (up is more) and pitch cues (low is large) to size interact, and whether similar biases influence size perception along the horizontal axis. In Experiment 1, we measured listeners’ voice-based judgments of human body size using pitch-manipulated voices projected from a high versus a low, and a right versus a left, spatial location. Listeners associated low spatial locations with largeness for lowered-pitch but not for raised-pitch voices, demonstrating that pitch overrode vertical-elevation cues. Listeners associated rightward spatial locations with largeness, regardless of voice pitch. In Experiment 2, listeners performed the task while sitting or standing, allowing us to examine self-referential cues to elevation in size estimation. Listeners associated vertically low and rightward spatial cues with largeness more for lowered- than for raised-pitch voices. These correspondences were robust to sex (of both the voice and the listener) and head elevation (standing or sitting); however, horizontal correspondences were amplified when participants stood. Moreover, when participants were standing, their judgments of how much larger men’s voices sounded than women’s increased when the voices were projected from the low speaker. Our results provide novel evidence for a multidimensional spatial mapping of pitch that is generalizable to human voices and that affects performance in an indirect, ecologically relevant spatial task (body size estimation). These findings suggest that crossmodal pitch correspondences evoke both low-level and higher-level cognitive processes

Guidelines for Disclosing Genetic Information to Family Members: from Development to Use

[À l'origine dans / Was originally part of : CRDP - Droit, biotechnologie et rapport au milieu