Deciphering the Microbiology of Respiratory Infection Using Precision Metagenomics Analysis

Carpenter, R., Sharma, R., Brown, E., Vine, T.
Publication date
October 2022
Abstracts, The Journal of Molecular Diagnostics
Elsevier Inc.
Introduction: Metagenomic sequencing using next-generation sequencing (NGS) technology has unparalleled power of comprehensive microbial profiling. NGS can detect unknown target organism(s) often undetected by routine microbiological and polymerase chain reaction (PCR) methods. We deployed a comprehensive NGS panel for simultaneous detection of 187 bacteria, 42 viruses, 53 fungi, and 1,218 antimicrobial resistance markers in 20 respiratory infection samples. NGS results were compared to a 36- target PCR panel commonly used for molecular diagnosis of respiratory infection.
Methods: DNA and RNA were extracted separately and we prepared libraries using the Illumina RNA Prep with Enrichment kit. Indexed libraries were enriched for microbial content by hybridization capture with the Respiratory Pathogen ID-AMR panel (Illumina). Final libraries were sequenced using the Illumina MiniSeq instrument to yield paired-end reads of 75 bp length. Sequencing data were analyzed by the IDbyDNA-Explify portal. A final explify report was generated containing the quantitative identification of viruses, bacteria, and fungi in each sample, including the antibiotic resistance marker. Same samples were also analyzed with a 36-target semi-quantitative PCR panel, and results obtained from both technologies were compared.