Distribution-based clustering: using ecology to refine the operational taxonomic unit.

16S rRNA sequencing, commonly used to survey microbial communities, begins by grouping individual reads into operational taxonomic units (OTUs). There are two major challenges in calling OTUs: identifying bacterial population boundaries and differentiating true diversity from sequencing errors. Current approaches to identifying taxonomic groups or eliminating sequencing errors rely on sequence data alone, but both of these activities could be informed by the distribution of sequences across samples. Here, we show that using the distribution of sequences across samples can help identify population boundaries even in noisy sequence data. The logic underlying our approach is that bacteria in different populations will often be highly correlated in their abundance across different samples. Conversely, 16S rRNA sequences derived from the same population, whether slightly different copies in the same organism, variation of the 16S rRNA gene within a population, or sequences generated randomly in error, will have the same underlying distribution across sampled environments. We present a simple OTU-calling algorithm (distribution-based clustering) that uses both genetic distance and the distribution of sequences across samples and demonstrate that it is more accurate than other methods at grouping reads into OTUs in a mock community. Distribution-based clustering also performs well on environmental samples: it is sensitive enough to differentiate between OTUs that differ by a single base pair yet predicts fewer overall OTUs than most other methods. The program can decrease the total number of OTUs with redundant information and improve the power of many downstream analyses to describe biologically relevant trends.

The Vaginal Microbiome as a Tool to Predict rASRM Stage of Disease in Endometriosis: a Pilot Study.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher's exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1-2 verses rASRM stages 3-4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present.

Using fecal microbiota as biomarkers for predictions of performance in the selective breeding process of pedigree broiler breeders.

Much work has been dedicated to identifying members of the microbial gut community that have potential to augment the growth rate of agricultural animals including chickens. Here, we assessed any correlations between the fecal microbiome, a proxy for the gut microbiome, and feed efficiency or weight gain at the pedigree chicken level, the highest tier of the production process. Because selective breeding is conducted at the pedigree level, our aim was to determine if microbiome profiles could be used to predict feed conversion or weight gain in order to improve selective breeding. Using 16s rRNA amplicon sequencing, we profiled the microbiomes of high and low weight gain (WG) birds and good and poor feed efficient (FE) birds in two pedigree lineages of broiler chickens. We also aimed to understand the dynamics of the microbiome with respect to maturation. A time series experiment was conducted, where fecal samples of chickens were collected at 6 points of the rearing process and the microbiome of these samples profiled. We identified OTUs differences at different taxonomic levels in the fecal community between high and low performing birds within each genetic line, indicating a specificity of the microbial community profiles correlated to performance factors. Using machine-learning methods, we built a classification model that could predict feed conversion performance from the fecal microbial community. With respect to maturation, we found that the fecal microbiome is dynamic in early life but stabilizes after 3 weeks of age independent of lineage. Our results indicate that the fecal microbiome profile can be used to predict feed conversion, but not weight gain in these pedigree lines. From the time series experiments, it appears that these predictions can be evaluated as early as 20 days of age. Our data also indicates that there is a genetic factor for the microbiome profile.

Inoculum composition determines microbial community and function in an anaerobic sequential batch reactor.

The sustainable recovery of resources from wastewater streams can provide many social and environmental benefits. A common strategy to recover valuable resources from wastewater is to harness the products of fermentation by complex microbial communities. In these fermentation bioreactors high microbial community diversity within the inoculum source is commonly assumed as sufficient for the selection of a functional microbial community. However, variability of the product profile obtained from these bioreactors is a persistent challenge in this field. In an attempt to address this variability, the impact of inoculum on the microbial community structure and function within the bioreactor was evaluated using controlled laboratory experiments. In the course of this work, sequential batch reactors were inoculated with three complex microbial inocula and the chemical and microbial compositions were monitored by HPLC and 16S rRNA amplicon analysis, respectively. Microbial community dynamics and chemical profiles were found to be distinct to initial inoculate and highly reproducible. Additionally we found that the generation of a complex volatile fatty acid profile was not specific to the diversity of the initial microbial inoculum. Our results suggest that the composition of the original inoculum predictably contributes to bioreactor community structure and function.

Profiling Living Bacteria Informs Preparation of Fecal Microbiota Transplantations.

Fecal microbiota transplantation is a compelling treatment for recurrent Clostridium difficile infections, with potential applications against other diseases associated with changes in gut microbiota. But variability in fecal bacterial communities-believed to be the therapeutic agent-can complicate or undermine treatment efficacy. To understand the effects of transplant preparation methods on living fecal microbial communities, we applied a DNA-sequencing method (PMA-seq) that uses propidium monoazide (PMA) to differentiate between living and dead fecal microbes, and we created an analysis pipeline to identify individual bacteria that change in abundance between samples. We found that oxygen exposure degraded fecal bacterial communities, whereas freeze-thaw cycles and lag time between donor defecation and transplant preparation had much smaller effects. Notably, the abundance of Faecalibacterium prausnitzii-an anti-inflammatory commensal bacterium whose absence is linked to inflammatory bowel disease-decreased with oxygen exposure. Our results indicate that some current practices for preparing microbiota transplant material adversely affect living fecal microbial content and highlight PMA-seq as a valuable tool to inform best practices and evaluate the suitability of clinical fecal material.

Computational methods for high-throughput comparative analyses of natural microbial communities.

One of the most widely employed methods in metagenomics is the amplification and sequencing of the highly conserved ribosomal RNA (rRNA) genes from organisms in complex microbial communities. rRNA surveys, typically using the 16S rRNA gene for prokaryotic identification, provide information about the total diversity and taxonomic affiliation of organisms present in a sample. Greatly enhanced by high-throughput sequencing, these surveys have uncovered the remarkable diversity of uncultured organisms and revealed unappreciated ecological roles ranging from nutrient cycling to human health. This chapter outlines the best practices for comparative analyses of microbial community surveys. We explain how to transform raw data into meaningful units for further analysis and discuss how to calculate sample diversity and community distance metrics. Finally, we outline how to find associations of species with specific metadata and true correlations between species from compositional data. We focus on data generated by next-generation sequencing platforms, using the Illumina platform as a test case, because of its widespread use especially among researchers just entering the field.