Ebola Molecular Inversion Probe Detection using Targeted Next-Generation Sequencing for Clinical Diagnostics

Next-generation sequencing is increasingly being used for diagnostic applications such as cancer and characterization of hereditary disease. The transition of this technology to agnostic infectious disease diagnostics is not... [ view full abstract ]

Next-generation sequencing is increasingly being used for diagnostic applications such as cancer and characterization of hereditary disease. The transition of this technology to agnostic infectious disease diagnostics is not as straightforward for several reasons. These reasons include a lack of specificity needed for FDA approval as well as the sequencing depth required to make a high confidence positive determination above host background sequences. To address the limitations of specificity and target coverage, we developed a filovirus-specific, targeted sequencing assay using molecular inversion probes (MIPs) coupled to amplicon identification by next-generation sequencing. Each MIP contains a conserved backbone with 3’ and 5’ primer binding sequences that are complementary to the target of interest, and these probe ends flank a specific target sequence. An enzymatic polymerization and ligation fill-in reaction captures the target sequence, and a subsequent PCR of the captured sequence allows identification by next-generation sequencing. The assay we designed here contains 68 different filovirus-specific MIPs in a single tube reaction. This assay positively detects Ebola virus, Sudan virus, Taï Forest virus, Bundibugyo virus, and Marburg virus. Thorough clinical performance assessments for Ebola virus and Bundibugyo virus were conducted using spike-in human serum samples for preliminary limit of detection, confirmation, inclusivity, and cross contamination (read bleeding). Clinical applicability is demonstrated with the positive detection of nonhuman primate Ebola virus samples and human Ebola and Bundibugyo virus clinical samples. Based on these data, we present a viable approach for targeted, broad pathogen detection using next-generation sequencing.