Deciphering the impact of contaminating microbiota in DNA extraction reagents on metagenomic next-generation sequencing workflows.

The widespread adoption of metagenomic next-generation sequencing has revolutionized microbial detection, yet contaminating DNA in laboratory reagents poses significant challenges for result interpretation. This study investigated microbial contamination profiles across four commercial DNA extraction reagent brands (M, Q, R, and Z) and assessed batch-to-batch variability. Extraction blanks were generated using molecular-grade water or ZymoBIOMICS Spike-in Control I as input materials. Analysis revealed distinct background microbiota profiles between brands, with some containing common pathogenic species that could affect clinical interpretation. Notably, background contamination patterns varied significantly between different lots of the same brand, highlighting the need for lot-specific microbiota profiling. Site-specific environmental contaminants were identified through analysis of 30 control samples from a single study site. Additionally, comparison of blood samples from healthy individuals with control samples suggested no evidence of a consistent blood microbiome, suggesting that "extraction blanks" may serve as negative controls in clinical metagenomic testing of sterile liquid biopsy samples. These findings emphasize the importance of including negative controls in every run and underscore the need for manufacturers to provide comprehensive background microbiota data for each reagent lot to optimize clinical interpretation and minimize false-positive results. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) has revolutionized pathogen detection and microbiome studies, but contamination from DNA extraction reagents remains a critical challenge. This study highlights the significant variability in background microbiota profiles across reagent brands and manufacturing lots, emphasizing the need for manufacturers to provide detailed contamination profiles. Our findings underscore the importance of implementing extraction blanks as standard controls and incorporating bioinformatics tools to account for background noise. These measures are essential to enhance the reliability of mNGS results and prevent diagnostic errors, particularly in clinical settings where contamination could mask or mimic pathogen signals. Additionally, our confirmation that healthy blood lacks a consistent microbiome helps streamline control selection in clinical testing protocols, potentially reducing costs and complexity in clinical mNGS workflows.