Dichloromethane biodegradation in multi-contaminated groundwater: Insights from biomolecular and compound-specific isotope analyses

International audienc

Marine microbial community structure assessed from combined metagenomic analysis and ribosomal amplicon deep-sequencing

The microbial taxonomic composition of the three domains of life in two coastal plankton samples was assessed by random total community metagenomic sequencing and PCR-based rDNA amplicon deep-sequencing in order to compare the resulting diversity and investigate possible limitations and complementarities of each method. The various universal primer sets, used to amplify different hypervariable rDNA regions, revealed the same major high-level taxonomic groups in Bacteria and unicellular Eukaryota, and showed a scarce Archaea apparent richness. However, significant differences were found between the different primer sets (p-value < 0.05, with the Kolmogorov–Smirnov test), regarding both operational taxonomic unit (OTU) richness and relative abundance of the major high-level taxonomic groups detected. Based on the metagenomic approach, the phylum Bacteroidetes dominated the prokaryotic community, followed by Proteobacteria, while the detected eukaryotic unicellular taxa belonged to the groups of Alveolata, Fungi, Chlorophyta, Stramenopiles and Phaeophyceae. These groups were found to carry genes typically found in microbial communities, which are linked to DNA, RNA and protein metabolism and the synthesis of nucleotides, amino acids, carbohydrates and vitamins. Although our findings suggest that the total community metagenomic approach can provide a more comprehensive picture of the planktonic microbial community structure, a number of issues associated with this approach emerged. These issues include the still relatively high cost compared to amplicon sequencing, the possible low coverage of the full marine diversity, the insufficiency of databases for other gene markers than the small subunit gene, and the bias towards bacterial sequences because of their higher abundance relative to eukaryotes in marine environments. © 2015 Taylor & Francis

Optimized and standardized 192-plex solution for 16S rDNA gene sequencing on Illumina Miseq platform to assess soil biodiversity

The growing need to survey the tremendous microbial diversity in a culture independent manner, has led to the development of molecular methods through sequence profiling of conserved genes such as 16S rDNA. Next-generation sequencing technologies are now used routinely to assess bacterial communities composition in complex environmental samples. Recently, the improvement of the Illumina MiSeq platform to a 2×300 bases paired-end version made it much more attractive for 16S rDNA amplicons sequencing enabling the analysis of a longer region thus facilitating taxonomic assignation. Additionally, the Illumina V3 chemistry enables high-throughput metagenomics analysis at the greatest coverage yet possible with a lower cost per sequence. Nevertheless, known limitations associated with the sequencing of low sequence diversity samples have hampered harnessing its true potential to sequence 16S rDNA gene amplicons. Moreover, validated Illumina 16S rDNA amplicon sequencing protocols do not support more than 96 samples per run, which underutilized the overall capacity of a sequencing run. We therefore worked on the development of a standardized and optimized high-multiplexed metagenomic solution for the exploration of complex environments. We developed an integrated solution combining several technical and bioinformatical strategies that are (i) a custom and standardized amplification protocol for 16S rDNA library preparation to minimize as full as possible technical biases; (ii) a custom 192-index strategy within a single run; (iii) the inclusion of a nucleotide sequence variability at the first sequencing cycles; (iv) an accurate paired-end reads assembly process for full length amplicon analysis and (v) a dedicated pipeline for taxonomic affiliation of 16S rDNA sequences and microbial communities analysis. Our solution validated within soil samples provides high sequencing accuracy and technical reproducibility with no index biases based on taxonomic distribution analysis. The association of technical and bioinformatical improvements yields substantial cost reductions and provides greater target flexibility to assess soil biodiversity as well as functions of microbial communities through carbon, phosphorus or nitrogen cycles in future developments

Dynamique des communautés bactériennes et fongiques de la rhizosphère au cours de l’infection de racines de Brassica rapa par le protiste obligatoire, agent de la hernie des crucifères, Plasmodiophora brassicae.

International audienc

PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification

International audienc

The Human Gut Chip "HuGChip\u27\u27, an explorative phylogenetic microarray for determining gut microbiome diversity at family level

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearson\u27s correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays

The human gut chip "HuGChip", an explorative phylogenetic microarray for determining gut microbiome diversity at family level.

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearson's correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays