A novel synthetic nucleic acid mixture for quantification of microbes by mNGS

Metagenomic next-generation sequencing (mNGS) provides considerable advantages in identifying emerging and re-emerging, difficult-to-detect and co-infected pathogens; however, the clinical application of mNGS remains limited primarily due to the lack of quantitative capabilities. This study introduces a novel approach, KingCreate-Quantification (KCQ) system, for quantitative analysis of microbes in clinical specimens by mNGS, which co-sequence the target DNA extracted from the specimens along with a set of synthetic dsDNA molecules used as Internal-Standard (IS). The assay facilitates the conversion of microbial reads into their copy numbers based on IS reads utilizing a mathematical model proposed in this study. The performance of KCQ was systemically evaluated using commercial mock microbes with varying IS input amounts, different proportions of human genomic DNA, and at varying amounts of sequence analysis data. Subsequently, KCQ was applied in microbial quantitation in 36 clinical specimens including blood, bronchoalveolar lavage fluid, cerebrospinal fluid and oropharyngeal swabs. A total of 477 microbe genetic fragments were screened using the bioinformatic system. Of these 83 fragments were quantitatively compared with digital droplet PCR (ddPCR), revealing a correlation coefficient of 0.97 between the quantitative results of KCQ and ddPCR. Our study demonstrated that KCQ presents a practical approach for the quantitative analysis of microbes by mNGS in clinical samples.