Deconvolution and Assembly of Metgenomes Using Chromatin Conformation Capture

DESCRIPTION provided by applicant The diversity of communities of microorganisms andquot microbiotaandquot in our bodies in soils and throughout all ecosystems known on Earth has only recently been realized Different microbiota are found at multiple body sites where some provide health benefits and others cause disease Analysis of these communities will reveal new ways to determine predisposition to diseases and will enable manipulation of the human microbiota to optimize human health Other microbiota are found in and around plant cells where some provide nutritional and anti disease benefits while others cause disease Understanding plant microbiota will identify and define new ecologically compatible and sustainable agricultural practices and enable agricultural expansion to currently unsuitable land Furthermore the unprecedented diversity of microorganisms throughout all ecosystems provides for tremendous and uncharted genetic diversity with far reaching industrial applications Technology Hurdle Much of the information we have about microbiota derives from high throughput sequencing technologies Because the vast majority of microorganisms are unknown and cannot be purified the microorganisms in microbiota must remain mixed and are co sequenced andquot metagenomicsandquot Using conventional methods it is difficult to decipher which sequences belong to specific microorganisms andquot deconvolutionandquot because information regarding the cell of origin is lost upon breaking cells and preparation of DNA for sequencing especially for complex genomes with multiple chromosomes or plasmids Our Technological Advance We used andquot chromosome conformation captureandquot to make both intra and inter chromosomal DNA crosslinks stable linkages prior to breaking cells and processing of DNA This allowed us to know which sequences originated in the same cell during deconvolution In our initial studies we successfully assembled artificially mixed populations of microorganisms fungal bacterial and archaeal species including those with complex genomes Hypothesis We hypothesize that our method can be applied to natural microbiota with unknown species at unknown concentrations Specific Aims To adapt wet lab chromosome conformation capture methods to real world metagenomic samples including difficult and low biomass samples and develop production quality software optimized for assembling low abundance genomes strain deconvolution and plasmid assignment Overall Impact Upon completion we believe our methods will become the standard for metagenomic sequencing to better realize the potential that discoveries of human plant and ecosystem microbiota have to offer PUBLIC HEALTH RELEVANCE Shotgun sequencing of mixed microbial populations yields large numbers of independent sequences that cannot be assembled into complete genomes due to lack of information on their cellular origin Our method connects these sequences into complete genomes and our proposal extends this technology to natural communities of microorganisms with unknown species and abundances We believe that this approach will become the technology standard in metagenomics enabling its much anticipated benefits to human health agriculture our environment and industry