CuReSim-LoRM: A Tool to Simulate Metabarcoding Long Reads.

Metabarcoding DNA sequencing has revolutionized the study of microbial communities. Third-generation sequencing producing long reads had opened up new perspectives. Obtaining the full-length ribosomal RNA gene would permit one to reach a better taxonomic resolution at the species or the strain level. However, Oxford Nanopore Technologies (ONT) sequencing produces reads with high error rates, which introduces biases in analysis. Understanding the biases introduced during the analysis allows one to better interpret the biological results and take care of conclusions drawn from metabarcoding experiments. To benchmark an analysis process, the ground truth, i.e., the real composition of the microbial community, has to be known. In addition to artificial mock communities, simulated data are often used to evaluate the biases and performances of the bioinformatics analysis step. Currently, no specific tool has been developed to simulate metabarcoding long reads, mimic the error rate and the length distribution, and allow one to benchmark the analysis process. Here, we introduce CuReSim-LoRM, for the customized read simulator to generate long reads for metabarcoding. We showed that CuReSim-LoRM is able to produce reads with varying error rates and length distributions by mimicking the real data very well.

Low dose dietary contamination with deoxynivalenol mycotoxin exacerbates enteritis and colorectal cancer in mice.

BACKGROUND: The mycotoxin deoxynivalenol (DON) is a frequent contaminant of grain and cereal products worldwide. Exposure to DON can cause gastrointestinal inflammation, disturb gut barrier function, and induce gut dysbiosis in vivo under basal conditions, but little is known about the effects of DON ingestion in individuals with pre-existing gastrointestinal disease. OBJECTIVES: Mice were orally exposed to 10 and 100 µg/kg bw/day of DON, corresponding to 10 to 100-fold human tolerable daily intake concentrations, and to the translation in mice of current human daily intake. The effects of DON exposure were explored under steady-state conditions, and in murine models of enteritis and colorectal cancer (CRC). RESULTS: After 8 days of DON exposure, an increase of histomorphological and molecular parameters of epithelial proliferation were observed in normal mice, from the duodenum to the colon. The same exposure in a murine model of indomethacin-induced enteritis led to exacerbation of lesion development and induction of ileal cytokines. DON exposure also worsened the development of colitis-associated CRC in mice as shown by increases in endoscopic and histological colitis scores, tumor grades, and histological hyperplasia. In colon of DON-exposed mice, upstream and downstream ERK signaling genes were upregulated including Mapk1, Mapk3, Map 2k1, Map2k2 core ERK pathway effectors, and Bcl2 and Bcl2l1 antiapoptotic genes. The effects observed in the CRC model were associated with alterations in cecal microbiota taxonomic composition and metabolism of bacterial fucose and rhamnose. Strong Spearman's correlations were revealed between the relative abundance of the changed bacterial genera and CRC-related variables. DISCUSSION: Ingestion of DON mycotoxin at concentrations representative of human real-world exposure worsened the development of indomethacin-induced enteritis and colitis-associated CRC in mice. Our results suggest that even at low doses, which are currently tolerated in the human diet, DON could promote the development of intestinal inflammatory diseases and CRC.

Murine in utero exposure to simulated complex urban air pollution disturbs offspring gut maturation and microbiota during intestinal suckling-to-weaning transition in a sex-dependent manner.

BACKGROUND: Emerging data indicate that prenatal exposure to air pollution may lead to higher susceptibility to several non-communicable diseases. Limited research has been conducted due to difficulties in modelling realistic air pollution exposure. In this study, pregnant mice were exposed from gestational day 10-17 to an atmosphere representative of a 2017 pollution event in Beijing, China. Intestinal homeostasis and microbiota were assessed in both male and female offspring during the suckling-to-weaning transition. RESULTS: Sex-specific differences were observed in progeny of gestationally-exposed mice. In utero exposed males exhibited decreased villus and crypt length, vacuolation abnormalities, and lower levels of tight junction protein ZO-1 in ileum. They showed an upregulation of absorptive cell markers and a downregulation of neonatal markers in colon. Cecum of in utero exposed male mice also presented a deeply unbalanced inflammatory pattern. By contrast, in utero exposed female mice displayed less severe intestinal alterations, but included dysregulated expression of Lgr5 in colon, Tjp1 in cecum, and Epcam, Car2 and Sis in ileum. Moreover, exposed female mice showed dysbiosis characterized by a decreased weighted UniFrac ß-diversity index, a higher abundance of Bacteroidales and Coriobacteriales orders, and a reduced Firmicutes/Bacteroidetes ratio. CONCLUSION: Prenatal realistic modelling of an urban air pollution event induced sex-specific precocious alterations of structural and immune intestinal development in mice.

Oral exposure to polyethylene microplastics alters gut morphology, immune response, and microbiota composition in mice.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 µm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 µg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

Exposure to atmospheric Ag, TiO2, Ti and SiO2 engineered nanoparticles modulates gut inflammatory response and microbiota in mice.

The development of nanotechnologies is leading to greater abundance of engineered nanoparticles (EN) in the environment, including in the atmospheric air. To date, it has been shown that the most prevalent EN found in the air are silver (Ag), titanium dioxide (TiO2), titanium (Ti), and silicon dioxide (SiO2). As the intestinal tract is increasingly recognized as a target for adverse effects induced by inhalation of air particles, the aim of this study was to assess the impact of these 4 atmospheric EN on intestinal inflammation and microbiota. We assessed the combined toxicity effects of Ag, Ti, TiO2, and SiO2 following a 28-day inhalation protocol in male and female mice. In distal and proximal colon, and in jejunum, EN mixture inhalation did not induce overt histological damage, but led to a significant modulation of inflammatory cytokine transcript abundance, including downregulation of Tnfα, Ifnγ, Il1ß, Il17a, Il22, IL10, and Cxcl1 mRNA levels in male jejunum. A dysbiosis was observed in cecal microbiota of male and female mice exposed to the EN mixture, characterized by sex-dependent modulations of specific bacterial taxa, as well as sex-independent decreased abundance of the Eggerthellaceae family. Under dextran sodium sulfate-induced inflammatory conditions, exposure to the EN mixture increased the development of colitis in both male and female mice. Moreover, the direct dose-response effects of individual and mixed EN on gut organoids was studied and Ag, TiO2, Ti, SiO2, and EN mixture were found to generate specific inflammatory responses in the intestinal epithelium. These results indicate that the 4 most prevalent atmospheric EN could have the ability to disturb intestinal homeostasis through direct modulation of cytokine expression in gut epithelium, and by altering the inflammatory response and microbiota composition following inhalation.

Erratum: Rémy et al. Modelling the Impact of Chronic Cigarette Smoke Exposure in Obese Mice: Metabolic, Pulmonary, Intestinal, and Cardiac Issues. Nutrients 2020, 12, 827.

The authors have requested that the following changes be made to their paper [...].

Rubrolone production by Dactylosporangium vinaceum: biosynthesis, modulation and possible biological function.

Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: • D. vinaceum encodes many BGC, but the majority are transcriptionally silent. • Chemical screening identifies molecules that modulate rubrolone production. • Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

Modelling the Impact of Chronic Cigarette Smoke Exposure in Obese Mice: Metabolic, Pulmonary, Intestinal, and Cardiac Issues.

Unhealthy lifestyle choices, such as bad eating behaviors and cigarette smoking, have major detrimental impacts on health. However, the inter-relations between obesity and smoking are still not fully understood. We thus developed an experimental model of high-fat diet-fed obese C57BL/6 male mice chronically exposed to cigarette smoke. Our study evaluated for the first time the resulting effects of the combined exposure to unhealthy diet and cigarette smoke on several metabolic, pulmonary, intestinal, and cardiac parameters. We showed that the chronic exposure to cigarette smoke modified the pattern of body fat distribution in favor of the visceral depots in obese mice, impaired the respiratory function, triggered pulmonary inflammation and emphysema, and was associated with gut microbiota dysbiosis, cardiac hypertrophy and myocardial fibrosis.

Comparative assessment of long-read error correction software applied to Nanopore RNA-sequencing data.

MOTIVATION: Nanopore long-read sequencing technology offers promising alternatives to high-throughput short read sequencing, especially in the context of RNA-sequencing. However this technology is currently hindered by high error rates in the output data that affect analyses such as the identification of isoforms, exon boundaries, open reading frames and creation of gene catalogues. Due to the novelty of such data, computational methods are still actively being developed and options for the error correction of Nanopore RNA-sequencing long reads remain limited. RESULTS: In this article, we evaluate the extent to which existing long-read DNA error correction methods are capable of correcting cDNA Nanopore reads. We provide an automatic and extensive benchmark tool that not only reports classical error correction metrics but also the effect of correction on gene families, isoform diversity, bias toward the major isoform and splice site detection. We find that long read error correction tools that were originally developed for DNA are also suitable for the correction of Nanopore RNA-sequencing data, especially in terms of increasing base pair accuracy. Yet investigators should be warned that the correction process perturbs gene family sizes and isoform diversity. This work provides guidelines on which (or whether) error correction tools should be used, depending on the application type. BENCHMARKING SOFTWARE: https://gitlab.com/leoisl/LR_EC_analyser.

Use of whole-genome sequencing in the molecular investigation of care-associated HCoV-OC43 infections in a hematopoietic stem cell transplant unit.

BACKGROUND: While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV-OC43 infections have rarely been investigated as healthcare-associated infections in this population. OBJECTIVES: In this report, HCoV-OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. STUDY DESIGN: Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient's sequences. Amino acids substitutions were analysed. RESULTS: Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. CONCLUSIONS: HCoV-OC43 may be implicated in healthcare-associated infections.

The Impact of Bioinformatics Pipelines on Microbiota Studies: Does the Analytical "Microscope" Affect the Biological Interpretation?

Targeted metagenomics is the solution of choice to reveal differential microbial profiles (defined by richness, diversity and composition) as part of case-control studies. It is well documented that each data processing step may have the potential to introduce bias in the results. However, selecting a bioinformatics pipeline to analyze high-throughput sequencing data from A to Z remains one of the critical considerations in a case-control microbiota study design. Consequently, the aim of this study was to assess whether the same biological conclusions regarding human gut microbiota composition and diversity could be reached using different bioinformatics pipelines. In this work, we considered four pipelines (mothur, QIIME, kraken and CLARK) with different versions and databases, and examined their impact on the outcome of metagenetic analysis of Ion Torrent 16S sequencing data. We re-analyzed a case-control study evaluating the impact of the colonization of the intestinal protozoa Blastocystis sp. on the human gut microbial profile. Although most pipelines reported the same trends in this case-control study, we demonstrated how the use of different pipelines affects the biological conclusions that can be drawn. Targeted metagenomics must therefore rather be considered as a profiling tool to obtain a broad sense of the variations of the microbiota, rather than an accurate identification tool.

A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.

Genome sequencing of virus has become a useful tool for better understanding of virus pathogenicity and epidemiological surveillance. Obtaining virus genome sequence directly from clinical samples is still a challenging task due to the low load of virus genetic material compared to the host DNA, and to the difficulty to get an accurate genome assembly. Here we introduce a complete sequencing and analyzing protocol called V-ASAP for Virus Amplicon Sequencing Assembly Pipeline. Our protocol is able to generate the viral dominant genome sequence starting from clinical samples. It is based on a multiplex PCR amplicon sequencing coupled with a reference-free analytical pipeline. This protocol was applied to 11 clinical samples infected with coronavirus OC43 (HcoV-OC43), and led to seven complete and two nearly complete genome assemblies. The protocol introduced here is shown to be robust, to produce a reliable sequence, and could be applied to other virus.

Chronic ingestion of deoxynivalenol at human dietary levels impairs intestinal homeostasis and gut microbiota in mice.

The mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereals and their by-products in areas with a moderate climate. Produced by Fusarium species, it is one of the most prevalent mycotoxins in cereal crops worldwide, and the most frequently occurring type B trichothecene in Europe. Due to its toxic properties, high stability and prevalence, the presence of DON in the food chain could represent a major public health risk. However, despite its well-known acute toxicological effects, information on the adverse effects of realistic exposure remains limited. We orally exposed mice during 9 months to DON at doses relevant for currently estimated human intake and explored the impact on various gut health parameters. DON exposure induced recruitment of regulatory B cells, and activation of regulatory T cells and dendritic cells in mesenteric lymph nodes. Several inflammatory parameters were increased in colon of DON-exposed mice, whereas inversely inflammatory markers were decreased in ileum. Histomorphological impairments were observed from the duodenum to the colon. Both colon and jejunum presented a hyperproliferation of epithelial cells and an increased expression of mature absorptive cells markers. Finally, DON exposure reshaped gut microbial structure and drastically disturbed the abundance of several bacterial phyla, families, and genera, leading to dysbiosis. Chronic oral exposure to human relevant doses of DON induces several disturbances of gut homeostasis with likely pathological implications for susceptible individuals.

Primary transcriptome analysis reveals importance of IS elements for the shaping of the transcriptional landscape of Bordetella pertussis.

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity. Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3' and 5'UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5'- or 3'-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

[Prospects for applications in human health of nanopore-based sequencing]. / Séquençage par nanopores - Perspectives d'applications en santé humaine.

High throughput sequencing has opened up new clinical opportunities moving towards a medicine of precision. Oncology, infectious diseases or human genomics, many applications have been developed in recent years. The introduction of a third generation of nanopore-based sequencing technology, addressing some of the weaknesses of the previous generation, heralds a new revolution. Portability, real time, long reads and marginal investment costs, these promising new technologies point to a new shift of paradigm. What are the perspectives opened up by nanopores for clinical applications?

MICRA: an automatic pipeline for fast characterization of microbial genomes from high-throughput sequencing data.

The increase in available sequence data has advanced the field of microbiology; however, making sense of these data without bioinformatics skills is still problematic. We describe MICRA, an automatic pipeline, available as a web interface, for microbial identification and characterization through reads analysis. MICRA uses iterative mapping against reference genomes to identify genes and variations. Additional modules allow prediction of antibiotic susceptibility and resistance and comparing the results of several samples. MICRA is fast, producing few false-positive annotations and variant calls compared to current methods, making it a tool of great interest for fully exploiting sequencing data.

Targeted metagenomic sequencing data of human gut microbiota associated with Blastocystis colonization.

In the past decade, metagenomics studies have become widespread due to the arrival of second-generation sequencing platforms characterized by low costs, high throughput and short read lengths. Today, although benchtop sequencers are considered to be accurate platforms to deliver data for targeted metagenomics studies, the limiting factor has become the analysis of these data. In a previous paper, we performed an Ion Torrent PGM 16S rDNA gene sequencing of faecal DNAs from 48 Blastocystis-colonized patients and 48 Blastocystis-negative subjects, in order to decipher the impact of this widespread protist on gut microbiota composition and diversity. We report here on the Ion Torrent targeted metagenomic sequencing and analysis of these 96 human faecal samples, and the complete datasets from raw to analysed data. We also provide the key steps of the bioinformatic analyses, from library preparation to data filtering and OTUs tables generation. This data represents a valuable resource for the scientific community, enabling re-processing of these targeted metagenomic datasets through various pipelines and a comparative evaluation of microbiota analysis methods.

Assessment of Common and Emerging Bioinformatics Pipelines for Targeted Metagenomics.

Targeted metagenomics, also known as metagenetics, is a high-throughput sequencing application focusing on a nucleotide target in a microbiome to describe its taxonomic content. A wide range of bioinformatics pipelines are available to analyze sequencing outputs, and the choice of an appropriate tool is crucial and not trivial. No standard evaluation method exists for estimating the accuracy of a pipeline for targeted metagenomics analyses. This article proposes an evaluation protocol containing real and simulated targeted metagenomics datasets, and adequate metrics allowing us to study the impact of different variables on the biological interpretation of results. This protocol was used to compare six different bioinformatics pipelines in the basic user context: Three common ones (mothur, QIIME and BMP) based on a clustering-first approach and three emerging ones (Kraken, CLARK and One Codex) using an assignment-first approach. This study surprisingly reveals that the effect of sequencing errors has a bigger impact on the results that choosing different amplified regions. Moreover, increasing sequencing throughput increases richness overestimation, even more so for microbiota of high complexity. Finally, the choice of the reference database has a bigger impact on richness estimation for clustering-first pipelines, and on correct taxa identification for assignment-first pipelines. Using emerging assignment-first pipelines is a valid approach for targeted metagenomics analyses, with a quality of results comparable to popular clustering-first pipelines, even with an error-prone sequencing technology like Ion Torrent. However, those pipelines are highly sensitive to the quality of databases and their annotations, which makes clustering-first pipelines still the only reliable approach for studying microbiomes that are not well described.

High-Throughput Sequencing, a VersatileWeapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology.

The recent progresses of high-throughput sequencing (HTS) technologies enable easy and cost-reduced access to whole genome sequencing (WGS) or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose.