Validation of massively parallel target enrichment approach

Sample processing and target enrichment are key steps in diagnostics and microbial genomic studies. However, until recently they received little attention in terms of development and optimization. This was partly because focusing on the detection and ecological aspects was much more exciting leading to a tangible result. However, it was also because diversity of sample matrix (water, food, blood, soil, manure, body fluids and tissues), the organisms to be detected (bacteria, protozoan, viruses, fungi), and the desired efficiency and uniformity of recovery (provides best detection limit, enrich target molecules, or maintain relative ratios) are too numerous and a universal solution was not expected. This proposal focuses on a massively parallel target enrichment approach employing baits to capture a large set of genes related to specific function and characterize the capture efficiency and gene abundance by high throughput sequencing. It also evaluates approaches leading to less than 60 minute assay (sample-to-result) to detect a set of organisms based on multiple genetic markers. A state-of-the-art approach is used to design 25,000 to 100,000 baits (120-mers) targeting 200 to 500 functional genes each for three different systems of varying complexity (antibiotic resistance gene panel, virulence and marker genes, and Escherichia coli populations). Samples already collected from relevant environment (dairy, watershed, wastewater treatment plant) are then subjected to the sample processing and enrichment protocol to capture the relevant functional gene microbiome and analyse it by 454 pyro-sequencing. In a point of care version of the assay, a subset of the antibiotic resistance gene is analysed by a portable system to assess the outcome by copy number obtained from isothermal amplification and compare it with the results obtained by pyro-sequencing. Both approaches are expected to enhance the capabilities necessary for functional metagenomic and multiplex diagnostics studies.