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

IntroductionProcess control for clinical metagenomic next-generation sequencing (mNGS) is not yet widely applied, while technical sources of bias are plentiful. If mNGS is to be safely used for clinical microbiology diagnostics, potential sources of error or variation in library preparation and sequencing need to be controlled for. AimWe aimed to develop an easy-to-use internal control (IC) method focussing on technical process control of library preparation and sequencing applied to mNGS in clinical microbiology diagnostics.MethodologyDNA of nine urine samples was sequenced on the Ion Torrent Proton and PGM in the absence and presence of Thermus thermophilus DNA as IC in incremental concentrations (0.5-2-5%). Between aliquots of each sample, we compared the IC relative abundance (RA), and after in silico subtraction of IC reads, the microbiota and the RA of pathogens. The proportion of cross-contaminant IC reads was determined by sequencing a replicate the absence of IC. The optimal IC spike-in concentration was defined as the lowest concentration still detectable in all samples with the smallest impact on the microbial community.ResultsThe RA of IC correlated linearly with the spiked IC concentration (r2=0.99). IC added in a concentration of 0.5% of total DNA concentration was detectable in all samples, regardless of human/bacterial composition and after in silico removal gave the smallest difference in RA of pathogens compared to the unspiked aliquot of the sample. Cross-contamination of IC reads was 0.001%. The microbiota of sample aliquots sequenced in the presence and absence of IC was highly similar after in silico removal of IC reads (median BC-dissimilarity per sample of 0.059), provided samples had sufficient bacterial read counts. ConclusionT. thermophilus DNA at a percentage of 0.5% of the total DNA concentration was successfully applied for the process control of mNGS of urine samples. We demonstrated negligible alterations in sample microbial composition after in silico subtraction of IC sequence reads. This approach contributes toward implementation of mNGS in the clinical microbiology laboratory.