Optimized bacterial DNA isolation method for microbiome analysis of human tissues

We present a bacterial DNA isolation method for human colon biopsies which includes reduction of human DNA and does not distort relative bacterial abundance at the phylum level. Shotgun sequencing resulted in 508/521 metagenomes (Shannon index; 2.9, UniFrac distance; 0.56). Fifteen colon biopsies were sequenced by both amplicon and shotgun sequencing resulting in similar bacterial profiles from phylum to genus level. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research. AbstractRecent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host-associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2 –5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DN A. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance ofFirmicutes,Bacteroidetes,γ-Proteobacteria, andActinobacteria assessed by qPCR. Our opti...

Source: MicrobiologyOpen - June 16, 2021 Category: Microbiology Authors: Carlijn E. Bruggeling, Daniel R. Garza, Soumia Achouiti, Wouter Mes, Bas E. Dutilh, Annemarie Boleij Tags: ORIGINAL ARTICLE Source Type: research