Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.