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

Metagenomic next generation sequencingNGSprovides a unique advantage to the discovery of disease causing pathogens as it does not rely on prior information about microbes and thus allows for the discovery new species which might otherwise have been missedA major limitation to the use of NGS in this context is the fact the host nucleic acid sequences vastly outnumber microbial DNA and RNAespecially when the infectious agent is in low abundanceThe premise of this proposal is to develop a method for highly efficient enrichment of viral and bacterial nucleic acids in biopsied samplesThis novel method substantially depletes the host genomic DNA and RNA from nucleic acid derived from cells or tissue and it efficiently concentrates the enriched microbial sequences for further analysisThe method utilizes hybridization to capture probes made up of a mixture of biotinylatedhuman tandem repeat oligosS andS rRNA probesand or oligo dTthat hybridize to the host DNA and RNA sequencesThe oligo hybridized host nucleic acid mixture is then separated from non captured sequences using an electrophoretic affinity separation method performed within a disposable cassetteThis device also accomplishes the separation and concentration of the non adsorbed nucleic acid by electroelutionThe process was successfully tested in preliminary workwherein andgtfold enrichment of doped bacterial DNA or viral RNA was achieved using a prototype cassette deviceAims of Phase I includeDetermination of the extended range of the enrichment process using human cellular nucleic acid doped with bacterialfungaland viral DNA and RNA in separate trialsFurther development of the electrophoretic cassette that accomplishes the affinity capture and concentration of the enriched nucleic acidUse the process to analyze nucleic acid isolated from actual autopsied brain and spinal fluid samples from encephalitis patientsalready in our possessionAnalysis of enriched neurological sample RNA and DNA using PGM Ion Torrent NGS sequencing to identify microbial sequences and determine if partial re assembly of the bacterial genomes is possible for the PGM dataConfigure a kit and specifications for the processThe products and methods resulting from this work will provide researchers with powerful tools for the discovery of new viruses and bacteria in patient samplesThe technology can ultimately be used in diagnostic determinations where disease causing microbes are either non abundantuncharacterizedor variants of known pathogens This work will create products for highly efficient enrichment of viral and bacterial nucleic acid in biopsied samplesThis technologycombined with next generation sequencing will open a door to the discovery of new disease causing pathogensalong with variants of known microbes that are causative or correlative of disease