The study highlights the significant impact of contaminating DNA in laboratory reagents and kits on sequence-based microbiome analyses, particularly in low-biomass samples. Contaminating DNA, which varies in composition between different kits and batches, can critically affect the results of both PCR-based 16S rRNA gene surveys and shotgun metagenomics. The authors demonstrate that contamination becomes dominant in sequence libraries as the input microbial biomass decreases, leading to misleading conclusions. They provide guidelines to mitigate contamination, including maximizing sample biomass, using negative controls, and monitoring reagent batches. The findings emphasize the need for caution in sequence-based microbiota studies to ensure accurate and reliable results.The study highlights the significant impact of contaminating DNA in laboratory reagents and kits on sequence-based microbiome analyses, particularly in low-biomass samples. Contaminating DNA, which varies in composition between different kits and batches, can critically affect the results of both PCR-based 16S rRNA gene surveys and shotgun metagenomics. The authors demonstrate that contamination becomes dominant in sequence libraries as the input microbial biomass decreases, leading to misleading conclusions. They provide guidelines to mitigate contamination, including maximizing sample biomass, using negative controls, and monitoring reagent batches. The findings emphasize the need for caution in sequence-based microbiota studies to ensure accurate and reliable results.