Bioconductor workflow for microbiome data analysis: from raw reads to community analyses [version 1; referees: 2 approved]

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut

Of mammals and bacteria in a rainforest: Temporal dynamics of soil bacteria in response to simulated N pulse from mammalian urine

Pulse-type perturbation through excreta by animals creates a mosaic of short-term high nutrient-load patches in the soil. How this affects microbial community composition and how long these impacts last are important for microbial community dynamics and nutrient cycling. Our study focused on the short-term responses to N by bacterial communities and ‘functional groups’ associated with the N cycle in a lowland evergreen tropical rainforest. We applied a single urea pulse, equivalent to urine-N deposition by medium-sized mammals to simulate N enrichment and changes in soil N availability, and analysed soil bacterial communities using molecular methods, before and after urea application. Urea addition increased mineral N availability and changed bacterial community composition, from phylum to operational taxonomic unit levels, however, taxon richness and diversity were unaffected. Taxa involved in the physiologically “narrow” processes of nitrification (e.g. Nitrosospira) and denitrification (e.g. Phyllobacteriaceae, Xanthomonadaceae and Comamonadaceae) increased their relative abundance, while N2-fixers (e.g. Rhodospirillales, and Rhizobiales) decreased after treatment. While a temporal legacy on both community composition and functional group profile was observable 58 and 159 days after treatment, at the latter date bacterial communities were already tending towards pre-treatment composition. We suggest that pulse-type perturbation by mammal urine that occurs on a daily basis has strong short-term effects on patch dynamics of soil microbiota and N availability. Such a spatio-temporally dynamic soil environment enhances overall microbial richness and diversity, and contributes to the apparent temporal resilience of community composition. A plain language summary is available for this article. © 2017 The Authors. Functional Ecology © 2017 British Ecological Societ

Temporal bacterial and metabolic development of the preterm gut reveals specific signatures in health and disease

Background - The preterm microbiome is crucial to gut health and may contribute to necrotising enterocolitis (NEC), which represents the most significant pathology affecting preterm infants. From a cohort of 318 infants, <32 weeks gestation, we selected 7 infants who developed NEC (defined rigorously) and 28 matched controls. We performed detailed temporal bacterial (n = 641) and metabolomic (n = 75) profiling of the gut microbiome throughout the disease. Results - A core community of Klebsiella, Escherichia, Staphyloccocus, and Enterococcus was present in all samples. Gut microbiota profiles grouped into six distinct clusters, termed preterm gut community types (PGCTs). Each PGCT reflected dominance by the core operational taxonomic units (OTUs), except of PGCT 6, which had high diversity and was dominant in bifidobacteria. While PGCTs 1–5 were present in infants prior to NEC diagnosis, PGCT 6 was comprised exclusively of healthy samples. NEC infants had significantly more PGCT transitions prior to diagnosis. Metabolomic profiling identified significant pathways associated with NEC onset, with metabolites involved in linoleate metabolism significantly associated with NEC diagnosis. Notably, metabolites associated with NEC were the lowest in PGCT 6. Conclusions - This is the first study to integrate sequence and metabolomic stool analysis in preterm neonates, demonstrating that NEC does not have a uniform microbial signature. However, a diverse gut microbiome with a high abundance of bifidobacteria may protect preterm infants from disease. These results may inform biomarker development and improve understanding of gut-mediated mechanisms of NEC

Diversity, structure and sources of bacterial communities in earthworm cocoons.

Animals start interactions with the bacteria that will constitute their microbiomes at embryonic stage. After mating, earthworms produce cocoons externally which will be colonized with bacteria from their parents and the environment. Due to the key role bacterial symbionts play on earthworm fitness, it is important to study bacterial colonization during cocoon formation. Here we describe the cocoon microbiome of the earthworms Eisenia andrei and E. fetida, which included 275 and 176 bacterial species, respectively. They were dominated by three vertically-transmitted symbionts, Microbacteriaceae, Verminephrobacter and Ca. Nephrothrix, which accounted for 88% and 66% of the sequences respectively. Verminephrobacter and Ca. Nephrothrix showed a high rate of sequence variation, suggesting that they could be biparentally acquired during mating. The other bacterial species inhabiting the cocoons came from the bedding, where they accounted for a small fraction of the diversity (27% and 7% of bacterial species for E. andrei and E. fetida bedding). Hence, earthworm cocoon microbiome includes a large fraction of the vertically-transmitted symbionts and a minor fraction, but more diverse, horizontally and non-randomly acquired from the environment. These data suggest that horizontally-transmitted bacteria to cocoons may play an important role in the adaptation of earthworms to new environments or diets

Localized Plasticity in the Streamlined Genomes of Vinyl Chloride Respiring Dehalococcoides

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism

A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology

The numerous species that make up the oral microbiome are now understood to play a key role in establishment and maintenance of oral health. The ability to taxonomically identify community members at the species level is important to elucidating its diversity and association to health and disease. We report the overall ecological effects of using a toothpaste containing enzymes and proteins compared to a control toothpaste on the plaque microbiome. The results reported here demonstrate that a toothpaste containing enzymes and proteins can augment natural salivary defences to promote an overall community shift resulting in an increase in bacteria associated with gum health and a concomitant decrease in those associated with periodontal disease. Statistical analysis shows significant increases in 12 taxa associated with gum health including Neisseria spp. and a significant decrease in 10 taxa associated with periodontal disease including Treponema spp. The results demonstrate that a toothpaste containing enzymes and proteins can significantly shift the ecology of the oral microbiome (at species level) resulting in a community with a stronger association to health

Unlocking preservation bias in the amber insect fossil record through experimental decay.

Fossils entombed in amber are a unique resource for reconstructing forest ecosystems, and resolving relationships of modern taxa. Such fossils are famous for their perfect, life-like appearance. However, preservation quality is vast with many sites showing only cuticular preservation, or no fossils. The taphonomic processes that control this range are largely unknown; as such, we know little about potential bias in this important record. Here we employ actualistic experiments, using, fruit flies and modern tree resin to determine whether resin type, gut microbiota, and dehydration prior to entombment affects decay. We used solid phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) to confirm distinct tree resin chemistry; gut microbiota of flies was modified using antibiotics and categorized though sequencing. Decay was assessed using phase contrast synchrotron tomography. Resin type demonstrates a significant control on decay rate. The composition of the gut microbiota was also influential, with minor changes in composition affecting decay rate. Dehydration prior to entombment, contrary to expectations, enhanced decay. Our analyses show that there is potential significant bias in the amber fossil record, especially between sites with different resin types where ecological completeness and preservational fidelity are likely affected