An Accurate Microbial DNA Quantification Assay for HTS Library Preparation of Human Biological Samples

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for multiple applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on the quantity of total DNA isolated. This method can overestimate the quantity of bacterial DNA due to the mixed nature of the sample, and consequently makes library preparation unreliable and variable. The purpose of this work was to develop an assay that can accurately and specifically quantify microbial DNA within a mixed sample for reliable 16S library preparation in advance of high throughput metagenomics sequencing. A qPCR method was optimized using universal 16s rDNA primers and cycling conditions adapted for qPCR. A commercially available microbial community DNA standard was used to develop an accurate, precise standard curve. Following qPCR optimization, 16S libraries from venous blood, saliva, semen, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS analyses were conducted with as low as 20 pg of microbial DNA as quantified using this method. Accurate quantification microbial DNA from DNA extracts resulted in consistent, successful library preparations for HTS analysis.