A Comparison of Homogenization vs. Enzymatic Lysis for Microbiome Profiling in Clinical Endoscopic Biopsy Tissue Samples

Abstract

Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla (Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract.

Keywords: DNA extraction; clinical biopsy; gastrointestinal tract; metagenomics; microbiome; quantitative PCR; whole genome sequencing.

Figure 1

qPCR quantifications of total bacterial content for all samples. The qPCR quantification of each experiment, including replicates, was plotted for each sample with different colors to distinguish two protocols. The readouts of replicates from the same sample are highly consistent. The control samples with known concentration also have been included in all experiments as the reference, demonstrating high accuracy for bacteria quantification. Six samples show significant difference between qPCR quantification of two protocols by student t-test (with red * mark).

Figure 2

Comparison of relative abundance of Fusobacterium sp. and three phyla for all samples. We assessed abundance of each of the above-mentioned bacterial clade relative to total 16S rDNA qPCR quantitation. Samples with significant difference between two protocols were marked with red *.

Figure 3

Comparison of bacteria identified from WGS data for five samples at genus level between two extraction methods. Both the homogenization method and the enzymatic lysis method can generate a similar set of microbial genus in all five samples (cosine similarity p < 0.05). Values represent percentage of bacteria reads found in each sample.