Thermus thermophilus DNA can be used as internal control for process monitoring of clinical metagenomic next-generation sequencing of urine samples.

Journal of Microbiological Methods
Victoria A. JanesC. Schultsz

Abstract

Metagenomics is increasingly considered for clinical diagnostics. In order for this technology to become integrated in the clinical microbiology laboratory, process controls are required. Molecular diagnostic tests typically integrate an internal control (IC) to detect potential sources of variation and technical failure. However, few studies report on the integration of an IC in metagenomics. We aimed to develop an easy-to-use IC method for the process control of library preparation and sequencing applied to metagenomics in clinical microbiology diagnostics using Thermus thermophilus DNA. DNA was extracted from urine samples and sequenced on the Ion Torrent Proton in the absence and presence of incremental concentrations (0.5-2-5%) of IC. Between aliquots of each sample, we compared the IC relative abundance (RA), and after in silico subtraction of IC reads, analysed microbial composition and the RA of pathogens. The optimal IC concentration was defined as the lowest concentration still detectable in all samples with the smallest impact on the microbial composition. The RA of IC correlated linearly with the spiked IC concentration (r2 = 0.99). IC added in a concentration of 0.5% of the total DNA concentration was detectable in...Continue Reading

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