Efficient Non-Biased Enrichment of Pathogen DNA and RNA in Biopsied Sample Nucleic Acid to Facilitate Novel Microbial Discovery

Metagenomic next generation sequencing (NGS) provides a unique advantage to the discovery ofdisease-causing pathogens as it does not rely on prior information about microbes and thus allows forthe discovery new species which might otherwise have been missed. A major limitation to the use ofNGS in this context is the fact the host nucleic acid sequences vastly outnumber microbial DNA andRNA, especially when the infectious agent is in low abundance. The premise of this proposal is todevelop a method for highly efficient enrichment of viral and bacterial nucleic acids in biopsied samples.This novel method substantially depletes the host genomic DNA and RNA from nucleic acid derived fromcells or tissue and it efficiently concentrates the enriched microbial sequences for further analysis. Themethod utilizes hybridization to capture probes made up of a mixture of biotinylated: human tandemrepeat oligos, 18S and 26S rRNA probes, and/or oligo(dT)20 that hybridize to the host DNA and RNAsequences. The oligo-hybridized host nucleic acid mixture is then separated from non-capturedsequences using an electrophoretic affinity separation method performed within a disposable cassette.This device also accomplishes the separation and concentration of the non-adsorbed nucleic acid byelectroelution. The process was successfully tested in preliminary work, wherein >1000-fold enrichmentof doped bacterial DNA or viral RNA was achieved using a prototype cassette device. Aims of Phase I include: 1) Determination of the extended range of the enrichment process usinghuman cellular nucleic acid doped with bacterial, fungal, and viral DNA and RNA in separate trials. 2)Further development of the electrophoretic cassette that accomplishes the affinity capture andconcentration of the enriched nucleic acid. 3) Use the process to analyze nucleic acid isolated fromactual autopsied brain and spinal fluid samples from encephalitis patients, already in our possession. 4)Analysis of enriched neurological sample RNA and DNA using PGM Ion Torrent NGS sequencing toidentify microbial sequences and determine if partial re-assembly of the bacterial genomes is possible forthe PGM data. 5) Configure a kit and specifications for the process. The products and methods resulting from this work will provide researchers with powerful tools forthe discovery of new viruses and bacteria in patient samples. The technology can ultimately be used indiagnostic determinations where disease causing microbes are either non-abundant, uncharacterized, orvariants of known pathogens.