Effects of Host Contamination in 16S rRNA Gene Sequencing of Low Biomass Samples

16S ribosomal RNA (rRNA) gene sequencing is a standard tool for microbial community analysis, yet it presents challenges, especially in low biomass samples where host DNA off-target amplification can occur. Such samples, with limited microbial DNA, amplify host genetic material during PCR, potentially distorting microbial profiles. This issue has been reported in several low-biomass studies. This analysis addresses the distortion of microbial profiles by amplifying host DNA in low-biomass samples. We investigate host contamination in 16S rRNA gene sequencing using a bacterial mock community dilution series spiked with varying amounts of human DNA. Off-target amplification dominated when bacterial mock content was less than 10% compared to human DNA, but total DNA concentration induced minimal bias. We found that post-clustering OTU filtering and reference genome mapping are effective methods for contamination mitigation.