Highly Sensitive Dual Amplicon-based NGS Approach for Bacterial Identification in Primary Clinical Specimens

Authors

• PP Khil
• J-H Youn
• J Ho
• JP Dekker
• KM Frank

Abstract

Next-generation sequencing (NGS) coupled with traditional amplicon-based methods holds promise for diagnostic characterization of pathogens in primary, uncultured, clinical specimens. Here we report the development of a highly sensitive, pan-bacterial diagnostic assay based on nested PCR amplification of the V1-V2 and V3-V4 regions of the 16S rRNA gene followed by sequencing using an Illumina MiSeq. Reads are analyzed with a newly designed modular pipeline that performs data pre-processing, OTU clustering and taxonomic assignment, species abundance estimation, and diagnostic report generation. Three improvements distinguish our assay from previously reported 16S-based approaches for use in clinical diagnostics: (1) nested PCR amplification combined with NGS, (2) semi-quantitative target concentration estimation using barcoded internal controls, and (3) analysis of four 16S hypervariable regions. For OTU load estimation, heptanucleotide barcodes were inserted into cloned Actinomadura nitritigenes 16S rDNA and these engineered targets were spiked at different concentrations. Initial analysis of the method including LoD studies using defined DNA mixes and isolate dilutions demonstrated reliable detection of < 100 copies of pathogen in the presence of 1000-fold excess of human cells. Initial clinical evaluation was performed with 24 primary uncultured urine samples representing a mixture of no growth, cultured urinary pathogens, and background commensal flora as reported using routine clinical culture. The method identified 110 organisms corresponding to commensal flora and all 12/12 urinary pathogens reported from culture in this set. Blinded validation of the method using 250 specimens is currently in progress under an NIH Clinical Center IRB protocol.

Scientific Focus Area: Microbiology and Infectious Diseases

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