Fig. 1

Design and workflow for targeted sequencing of the cervicovaginal microbiome. A The design and in silico validation of targeted sequencing involved the initial selection of microbial species from the CVM and their regions of interest (ROIs) within the 16S rRNA gene. Thereafter, single-molecule molecular inversion probes (smMIPs) were designed and validated to target specifically all the ROIs, which resulted in thousands of promising smMIPs. By performing in silico analyses, the amount of smMIPs were shortened and validated to profile all the microbial species, which resulted in the final selection of 30 different smMIPs that are able to achieve high-resolution microbiome profiling. B For CVM profiling, these 30 smMIPs are available to hybridize to the 16S rRNA gene of microbes (e.g., DNA or cDNA from RNA) identified as part of the cervicovaginal microbiome. In the cervicovaginal microbiome, hundreds of microbial species can be detected, playing a role in health and disease. smMIPs were selected based on extension and ligation arms that are shared between species and flanking hypervariable ROI that are unique per species. After smMIP hybridization and filling in the ROI gaps, followed by ligation, the library of circularized smMIPs is PCR amplified with barcoded Illumina primers and sequenced. All collected ROI sequences in a sample are then compared to a reference database containing reference ROIs from all microbial species of interest. Based on a combination of two or more ROIs, the microbiome can be annotated in high-resolution. The assay is made quantitative by incorporating a unique molecule identifier (UMI), which eliminated PCR amplification bias