Group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
Human Microbiome research team
Centro Superior de Investigación en Salud Pública (CSISP)
Avda. Cataluña 21, 46020 Valencia (SPAIN)
Contact: Alex Mira (Oral metagenome), M. Pilar Francino (Gut metagenome)
METAGENOMICS OF THE HUMAN-ASSOCIATED MICROBIOTA
We aim to evaluate and develop new approaches for studying the human microbiome that are not based on DNA cloning or PCR. This would avoid the biases introduced by these two approaches and would allow for the detection of bacterial species that would otherwise be under-represented or absent from current studies of the human microbiome. We believe that the new sequencing technologies make this possible and that such an unbiased approach will allow for a precise quantification of the different components of a microbiome. Amplification of the 16S rRNA gene by so-called "universal" primers followed by cloning in E coli plasmids to carry out sequencing has been the standard procedure for determination of the bacterial biodiversity in a sample, in spite of well known shortcomings such as the substantial cost, time and training required and the biases that make interpretation of the results difficult. Moreover, recent advances in next generation sequencing technologies have mainly been used to increase the number of 16S rRNA reads for sequencing but maintaining the biased PCR amplification step. As an alternative to PCR of 16S rRNA, we propose direct pyrosequencing of DNA (where 1% of the reads will have phylogenetic value) or cDNA (where 85% of the reads will presumably be ribosomal) without amplification and obviating cloning. Direct DNA pyrosequencing will provide, not only rRNA, but also conserved house-keeping gene sequences that can be used for phylogenetic identification and that, being single-copy, reflect more directly the relative frequency of different species in the sample. We are evaluating and comparing the validity of DNA and cDNA pyrosequencing approaches to accurately reflect the real biodiversity of a sample and the relative frequencies of the sample components. To this aim, we have tested in an "artificial" metagenome whether the frequencies of the obtained DNA and cDNA reads correlate with the known proportions of different bacteria used to generate the sample. Regarding functional studies, the advent of shotgun, cloning-based metagenomics techniques has improved the study of the human microbiome because it allows for the direct cloning of genome segments from all the species present obviating the need for culture. However, depending on the vector used (generally E. coli) and the genes to clone, certain genomic regions are rarely cloned successfully, for example because they are toxic to the surrogate host. As an alternative to metagenomic libraries, we are testing if the 400 bp-long pyrosequences now available through high throughput “next generation” sequencing technologies are sufficient to adscribe each read to a protein category, can provide sufficient functional information about the genes present and are also indicative of the initial frequency of those genes in the sample, obviating the need for cloning and opening a new, quantitative dimension in metagenomics. If these approaches are successful, they could be the end of cloning for many projects in microbiology and the beginning of a new model for exploratory microbiology of the human microbiota that is not dependent exclusively on ribosomal genes, that is quantitative, that requires less time and money, that is unbiased and that dedicates less time to technical and laboratory aspects and more time to the analysis of data that integrate the real biodiversity of complex samples. We are applying the new techniques to study the development of the human gastrointestinal microbiota in infants and the biodiversity of the supragingival dental plaque.