Critical assessment of intratumor and low-biomass microbiome using long-read sequencing.

The detection of low-biomass microbial DNA in human tissues is often confounded by contamination, as demonstrated in the debates over the existence of microbiomes in the placenta, brain, blood, and tumors. Here we show that genomic DNA fragment length serves as a discriminator: while genuine microbiome genomes have long genomic DNA fragments, contaminant DNA is typically short. Using germ-free mouse tissues with bacterial spike-ins and human cell lines, we developed a metric that normalizes microbial read length to host read length. Across multiple human tumor and normal tissues, we found genuine microbiome signals are largely limited to tissues with natural microbial exposure (e.g., gastrointestinal tract, cervix, vagina, skin), while other tissues (e.g. kidney, brain, blood, and placenta) showed no evidence of resident microbiome. These findings support DNA fragment length as a metric for quality controlling low-biomass microbiome profiling, clarifying the debates and strengthen future studies of resident microbiome in tissues.