Correlation between Targeted qPCR Assays and Untargeted DNA Shotgun Metagenomic Sequencing for Assessing the Fecal Microbiota in Dogs

SIMPLE SUMMARY: Untargeted shotgun DNA sequencing of fecal samples is a relatively novel approach to study the microbiome. This method allows better classification of bacteria on a species level compared to traditional 16S rRNA gene sequencing, and provides data about viruses, fungi, archaea, and functional genes. A targeted qPCR-based dysbiosis index has been recently introduced to evaluate the fecal microbiota in dogs. This study evaluated the agreement for core bacterial taxa between targeted qPCR assays and relative abundances obtained by shotgun DNA sequencing. We analyzed the fecal microbiota of 296 dogs with various clinical phenotypes using both methods. Significant correlations were found between the two methods, and the qPCR-based dysbiosis index accurately reflected shifts in the microbiome of dogs as observed by DNA shotgun sequencing. ABSTRACT: DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.