摘要The high host genetic background of tissue biopsies hinders the application of shotgun metagenomic sequencing in characterizing the tissue microbiota.We proposed an optimized method that removed host DNA from colon biopsies and examined the effect on metagenomic analysis.Human or mouse colon biopsies were divided into two groups,with one group undergoing host DNA depletion and the other serving as the control.Host DNAs were removed through differential lysis of mammalian and bacterial cells before sequencing.The impact of host DNA depletion on microbiota was compared based on phylogenetic diversity analyses and regression analyses.Removing host DNA enhanced bacterial sequencing depth and improved species discovery,increas-ing bacterial reads by 2.46±0.20 fold while reducing host reads by 6.80％±1.06％.Moreover,3.40 times more of bacterial species were detected after host DNA depletion.This was confirmed from mouse colon tissues,increasing bacterial reads by 5.46±0.42 fold while decreasing host reads by 10.2％±0.83％.Similarly,significantly more species were detected in the mouse colon tissue upon host DNA depletion(P＜0.001).Furthermore,an increased microbial richness was evident in the host DNA-depleted samples compared with non-depleted controls in human colon biopsies and mouse colon tissues(P＜0.001).Our optimized method of host DNA depletion improved the sensitivity of shotgun metagenomic sequencing in bacterial detection in the biopsy,which may yield a more accurate taxonomic profile of the tissue microbiota and identify bacteria that are important for disease initiation or progression.