Sensitive NGS method for the analysis of microbial and viral nucleic acid in cell free RNA

Doug Amorese 1,
1NuGEN Technologies, United States; 

Finding the causative agent of an infection can be challeng- ing. Low RNA quantity, poor quality, low viral titers, and high background from the host negatively impact viral detection from biofluids (blood, nasal swabs, CSF). PCR can be an effective means of detecting specific, well characterized organisms but only if primers are closely spaced and those organisms are represented in the test panel. RNA-Seq is an alternative unbiased method that has previ- ously been used with cfRNA to identify unknown infections in an impartial manner. However, this method typically requires higher RNA input and quality than typically exist in clinical samples. Fur- ther, the low titers of the pathogen RNA relative to the host and non- pathogen RNA necessitate deep sequencing of the libraries. Several reports have described the use of NuGEN’s isothermal amplification technology (SPIA) to overcome the low yields and poor RNA qual- ity for the detection of viruses in human biological fluids but these methods are still burdened with uninformative host transcripts.
Here we describe a single workflow that combines NuGEN’s; SPIA amplification technology, enzymatic fragmentation, and Any- Deplete (InDA-C targeted depletion method) to study the causative agent(s) and host response in nasal samples obtained from asth- matic and non-asthmatic children with presumptive respiratory infections. This unbiased method enabled efficient viral detection and detection of lowly expressed human transcripts with <1 million reads per sample. Data illustrating the efficiency of the depletion method and the coverage of the viral genomes will be presented.

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