SBIR/STTR Award attributes
Abstract The Specific Aim of this Phase II SBIR proposal is to develop an affordable commercial quality product for improved metagenomic sequencing and culture free microbial discoveryWith development of high throughput sequencing technologieswe now have the capacity to sequence entire genomes of cultured microorganismsHoweverwe only have a limited capacity to sequence pooled genomesmicrobiomesthrough metagenomic sequencingwhich includes sequencing the genomes of microorganisms that currently cannot be culturedUsing conventional protocolsthe association of DNA fragments from the same species is lost during the DNA preparation processcell lysisDNA purification and shearingIt is therefore near impossible to assign any specific DNA sequence to its origin without relying on a priori knowledgeTo overcome this hurdlewe have adapted the Hi C proximity ligation tool to join DNA molecules that are physically proximal to one another within an intact cellOur successful Phase I studies showed the feasibility of developing a commercial product for the construction of high quality Hi C libraries from fecalsoiland clinical samplesDeep sequencing of these DNA junctions enables us to reconstruct complete or near complete genomes and deconvolute mixed strains without any culturingWe have successfully applied this technology for deconvolution and assembly of artificially mixed populations of microorganisms that included various fungalbacterialand archaeal species as well as a number of real world metagenomic samplesThe method has proven to be highly accurate and efficientincluding associating multiple chromosomes and plasmids with their host microorganismWhile feasiblein order to become a commercial product we must develop our laboratory methods into commercial quality high throughput assay kits and finalize software developmentTo achieve our Specific Aimwe will carry out the following TasksTaskOptimize current Hi C kit protocolsTaskDeveloptest and manufacturewell metagenomic Hi C kitsTaskAlgorithm development and software optimization for analyzing Hi C dataTaskProduce a customer facing website used for data analysisCriteria for SuccessA successful product would have to reduce the laboratory workflow to underhours of prep timebe simple enough to allow multiplexingand bring our cost of goods down to $per sample or lessThe method would need to work on a wide array of metagenomic sample typessuch as fecalclinicaland environmental samples containing a variety of diverse microbesOur kit will consist of enzymes and buffers to generate an Illumina compatible Hi C library from a raw sample such asgrams of soil oruL of fecal material with a andltfailure rate by users with moderate levels of experienceAt least ten clients must independently validate our kitsThe accompanying software will be able to assemble known genomes with at leastaccuracy and generate at leastnovel genomes per sample with andgtcompleteness and andltcontamination as measured by the CheckM tool Narrative Our bodies and the environment are dominated by communities of microorganismsmicrobiotathat have profound effects on our healthagricultureenvironmentand diverse industrial processesHoweverwe only have a limited capacity to sequence pooled genomesmicrobiomeswhich includes sequencing the genomes of microorganisms that currently cannot be culturedMetagenomic sequencing has already proven itself highly usefulespecially in the areas of health benefits and human diseasesIt has provided insight into plant microbiomeswith implications for enhancing ecologically friendly agricultural managementand it has provided access to genomes of uncharted microorganisms that can be exploited for industrial processesThis is a proposal to overcome current limitations in sequencing that will allow any reasonably well trained molecular biologist to sequence even the most complex microbiomes with state of the art results
