We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.

/pdf/dada2-high-resolution-sample-inference-from-illumina-ygkdeyoxho.pdf

DADA2: High-resolution sample inference from Illumina amplicon data

Preface to the Princeton Landmarks in Biology Edition vii Preface xi Symbols Used xiii 1. The Importance of Islands 3 2. Area and Number of Speicies 8 3. Further Explanations of the Area-Diversity Pattern 19 4. The Strategy of Colonization 68 5. Invasibility and the Variable Niche 94 6. Stepping Stones and Biotic Exchange 123 7. Evolutionary Changes Following Colonization 145 8. Prospect 181 Glossary 185 References 193 Index 201

The Theory of Island Biogeography

Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

UPARSE: highly accurate OTU sequences from microbial amplicon reads

Background The analysis of microbial communities through DNA sequencing brings many challenges: the integration of different types of data with methods from ecology, genetics, phylogenetics, multivariate statistics, visualization and testing. With the increased breadth of experimental designs now being pursued, project-specific statistical analyses are often needed, and these analyses are often difficult (or impossible) for peer researchers to independently reproduce. The vast majority of the requisite tools for performing these analyses reproducibly are already implemented in R and its extensions (packages), but with limited support for high throughput microbiome census data. Results Here we describe a software project, phyloseq, dedicated to the object-oriented representation and analysis of microbiome census data in R. It supports importing data from a variety of common formats, as well as many analysis techniques. These include calibration, filtering, subsetting, agglomeration, multi-table comparisons, diversity analysis, parallelized Fast UniFrac, ordination methods, and production of publication-quality graphics; all in a manner that is easy to document, share, and modify. We show how to apply functions from other R packages to phyloseq-represented data, illustrating the availability of a large number of open source analysis techniques. We discuss the use of phyloseq with tools for reproducible research, a practice common in other fields but still rare in the analysis of highly parallel microbiome census data. We have made available all of the materials necessary to completely reproduce the analysis and figures included in this article, an example of best practices for reproducible research. Conclusions The phyloseq project for R is a new open-source software package, freely available on the web from both GitHub and Bioconductor.

/pdf/phyloseq-an-r-package-for-reproducible-interactive-analysis-1ez1xx3dlr.pdf

phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.

The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

We read with interest the study by Clooney et al, which identified determinants of microbiome variance in inflammatory bowel disease (IBD). Despite Crohn’s disease (CD) explaining most variance, most of the faecal microbiota variation was unexplained as previously described on population level. CD affects the entire gut, but most studies use stool samples as a proxy for the colonic environment. The small intestinal microbiome differs from stool and both vertical (proximal to distal) and horizontal (luminal to mucosal) gradients exist, but is generally understudied, especially in CD. Recently, we showed differences between the duodenal microbiota of healthy volunteers (HV) and functional dyspepsia (FD) patients, which may play a role in the pathogenesis of FD. Both aseptic duodenal and ileal samples (brush and biopsy) were collected during concurrent upper and lower endoscopy in patients with CD (see online supplemental methods). History of intestinal surgery, use of acid (proton pump inhibitors, PPIs) or immunosuppression (IS) and presence of macroscopic (erosions/ ulcers) and/or microscopic inflammation were recorded. Alpha diversity was compared between sampling types (biopsy/brush), groups and treatments (PPI/IS). Genera abundance profiles were compared along vertical (duodenum/ ileum) and horizontal (brush/biopsy) gradients with correction for multiple testing (false discovery rate (FDR) <0.1). Microbiota covariates were studied using distancebased redundancy analysis (genuslevel Aitchison distance) after centred logratio transformation. A total of 21 patients with CD (median (IQR) age 40 (26.5–50) years and body mass index (BMI) 23.5 (21.4–27.6) kg/m2, 52% female) and 30 HV (age 27 (24–33.5) years and BMI 23 (20–25.3) kg/m, 70% female) were included. Mucusassociated α-diversity (brushes) was higher in patients with CD versus controls (figure 1A) and lower with PPI in all subjects (p<0.05). Epitheliumassociated α-diversity (biopsies) was higher with IS in patients with CD (figure 1B). Duodenal α-diversity of brushes was lower than biopsies, and ileal α-diversity was similar (see online supplemental results). However, biopsies were likely contaminated as previously shown and also illustrated in the genera abundance profiles (top 10) across vertical and horizontal gradients (online supplemental figure 1). Within CD, sampling location (duodenum or ileum) but not demographics, history of surgery, treatment or presence of inflammation was a significant covariate of the mucusassociated microbiota variation of the small intestine (table 1). Using paired analyses, significant differences between duodenal and ileal brushes of patients with CD were found for 24 genera (online supplemental figure 2), of which the most common genus was Streptococcus, with a higher prevalence in the duodenum versus ileum of patients with CD (FDR<0.1). Interestingly, similar results were previously found in the colon of ulcerative colitis patients, with increased α-diversity in the mucus gel layer versus controls. Also, no effects of inflammation were found in colonic luminal or mucosal samples, which contrasts with changes in the faecal microbiome indicative of disease activity reported by Clooney et al. Although possibly more conserved in comparison to faecal microbiota, longitudinal sampling is needed to study the intraindividual variance of the small intestinal microbiome in relation to inflammation and treatment, including the effects of IS on mucosal diversity as previously described. Moreover, studies from different geographical regions are needed to investigate potential covariates, including ethnicity and diet, as demonstrated in the faecal microbiome. In conclusion, we showed significant small intestinal microbiota variation along the vertical and horizontal gradients in CD. The α-diversity of duodenal brush samples was highest in patients with Letter

Microbiome variance of the small bowel in Crohn’s disease

P095 Anti-inflammatory effect of high acetate concentration on organoid-derived epithelial monolayer from patients with Ulcerative Colitis

Tu1485: EXPOSURE TO MICROBIOTA FROM ULCERATIVE COLITIS PATIENTS IS INDUCING STRESS RESPONSES IN EPITHELIAL CELLS

Cellulose can be isolated from various raw materials and agricultural side streams and might help to reduce the dietary fiber gap in our diets. However, the physiological benefits of cellulose upon ingestion are limited beyond providing fecal bulk. It is barely fermented by the microbiota in the human colon due to its crystalline character and high degree of polymerization. These properties make cellulose inaccessible to microbial cellulolytic enzymes in the colon. In this study, amorphized and depolymerized cellulose samples with an average degree of polymerization of less than 100 anhydroglucose units and a crystallinity index below 30% were made from microcrystalline cellulose using mechanical treatment and acid hydrolysis. This amorphized and depolymerized cellulose showed enhanced digestibility by a cellulase enzyme blend. Furthermore, the samples were fermented more extensively in batch fermentations using pooled human fecal microbiota, with minimal fermentation degrees up to 45% and a more than eight-fold increase in short-chain fatty acid production. While this enhanced fermentation turned out to be highly dependent on the microbial composition of the fecal pool, the potential of engineering cellulose properties to increased physiological benefit was demonstrated.

https://www.mdpi.com/2304-8158/12/5/1100/pdf?version=1677923294

Decreasing the Crystallinity and Degree of Polymerization of Cellulose Increases Its Susceptibility to Enzymatic Hydrolysis and Fermentation by Colon Microbiota

Aim: The role of the human gut microbiome in colorectal cancer (CRC) is unclear as most studies on the topic are unable to discern correlation from causation. We apply two-sample Mendelian randomization (MR) to estimate the causal relationship between the gut microbiome and CRC. Materials and methods: We used summary-level data from independent genome-wide association studies to estimate the causal effect of 14 microbial traits (n=3,890 individuals) on overall CRC (55,168 cases, 65,160 controls) and site-specific CRC risk, conducting several sensitivity analyses to understand the nature of results. Results: Initial MR analysis suggested that a higher abundance of Bifidobacterium and presence of an unclassified group of bacteria within the Bacteroidales order in the gut increased overall and site-specific CRC risk. However, sensitivity analyses suggested that instruments used to estimate relationships were likely complex and involved in many potential horizontal pleiotropic pathways, demonstrating that caution is needed when interpreting MR analyses with gut microbiome exposures. In assessing reverse causality, we did not find strong evidence that CRC causally affected these microbial traits. Conclusions: Whilst our study initially identified potential causal roles for two microbial traits in CRC, importantly, further exploration of these relationships highlighted that these were unlikely to reflect causality.

/pdf/exploring-the-causal-role-of-the-human-gut-microbiome-in-2otnsu6s.pdf

Jeroen Raes

Papers

Microbiome variance of the small bowel in Crohn’s disease

P095 Anti-inflammatory effect of high acetate concentration on organoid-derived epithelial monolayer from patients with Ulcerative Colitis

Tu1485: EXPOSURE TO MICROBIOTA FROM ULCERATIVE COLITIS PATIENTS IS INDUCING STRESS RESPONSES IN EPITHELIAL CELLS

Decreasing the Crystallinity and Degree of Polymerization of Cellulose Increases Its Susceptibility to Enzymatic Hydrolysis and Fermentation by Colon Microbiota

Exploring the causal role of the human gut microbiome in colorectal cancer: Application of Mendelian randomization