摘要：SummaryFew validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.Graphical abstractDisplay OmittedHighlights•Lysis buffer keeps the microbial composition stable during various storage conditions•Host DNA depletion introduces a larger bias toward Gram-negative taxa•The HostZERO Microbial DNA kit performed best in human DNA depletion for metagenomics•Body site-specific approach based on microbial composition is needed to minimize biasBody substance sample; Microbiology; Omics
