Abstract
Recent 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 DNA. 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 of Firmicutes, Bacteroidetes, γ‐Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.
Highlights
The rapidly growing field of microbiome research is steadily revealing the role of the microbiome in human health and diseases
To address the current limitations in obtaining bacterial DNA from gut tissue samples that is suitable for shotgun metagenomic sequencing, here we present an optimized DNA isolation method
The significantly lower DNA yield after DNAse treatment was associated with an increased bacterial signal in qPCR (p = 0.004) (Figure 3a), which is indicative of a greater bacterial-to-human DNA fraction in the tissue DNA isolate and suggests bacterial DNA enrichment
Summary
The rapidly growing field of microbiome research is steadily revealing the role of the microbiome in human health and diseases. Shotgun metagenomic sequencing allows analysis of all sequences in the DNA isolate, resulting in an increased species detection with higher accuracy (Ranjan et al, 2016) Another major advantage of this technique is the ability to discriminate between microbial species and analyze their gene content including potential virulence factors (Ranjan et al, 2016). Our optimized protocol enriches the bacterial content of biopsies ranging from ~2–5 mm and was validated in the context of two large prospective studies on in vivo acquired tissue biopsies using shotgun metagenomics This method will contribute to reproducible research in the field of bacterial microbiome composition and function and will be of value for gut-related tissue and for those tissues where bacteria are underrepresented
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