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

Macconnell Research Corporation, San Diego CA

Investigators

Abstract

Metagenomic next generation sequencing (NGS) provides 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 missed. A major limitation to the use of NGS in this context is the fact the host nucleic acid sequences vastly outnumber microbial DNA and RNA, especially when the infectious agent is in low abundance. The premise of this proposal is to develop 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 from cells or tissue and it efficiently concentrates the enriched microbial sequences for further analysis. The method utilizes hybridization to capture probes made up of a mixture of biotinylated: human tandem repeat oligos, 18S and 26S rRNA probes, and/or oligo(dT)20 that hybridize to the host DNA and RNA sequences. The oligo-hybridized host nucleic acid mixture is then separated from non-captured sequences 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 by electroelution. The process was successfully tested in preliminary work, wherein >1000-fold enrichment of 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 using human 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 and concentration of the enriched nucleic acid. 3) Use the process to analyze nucleic acid isolated from actual 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 to identify microbial sequences and determine if partial re-assembly of the bacterial genomes is possible for the 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 for the discovery of new viruses and bacteria in patient samples. The technology can ultimately be used in diagnostic determinations where disease causing microbes are either non-abundant, uncharacterized, or variants of known pathogens.