Unraveling the significance of epithelial-associated bacteria in gastrointestinal diseases: Importance of choosing an optimal DNA extraction method

Understanding the significance of epithelial-associated bacteria in gastrointestinal diseases is essential for gaining insights into the complex host-microbiota interactions that influence disease development and progression. However, sequencing approaches face limitations due to the overwhelming presence of host DNA in the samples. PCR-based approaches like 16S rRNA gene amplicon sequencing enables taxonomic profiling and has been studied extensively in connection with a wide range of diseases and while still being a valuable method, microbiome research is moving towards metagenomic sequencing. This allows for the deciphering of both the bacterial community structure at a higher taxonomic resolution as well as insight into its functional potential. Here, we present a comparative study of three different DNA extraction methods for human colon biopsies: two commercially available kits (Qiagen Blood and tissue kit and Molzym ultra-deep microbiome prep) and one published optimized method (Saponin approach). The three methods were evaluated in terms of the ratio between host and bacterial DNA and their ability to retain the relative bacterial abundance at different taxonomic levels. Six colon biopsies (18 technical replicates) were sequenced with 16S rRNA gene amplicon and metagenomic sequencing resulting in distinct bacterial profiles dependent on the applied extraction method. The Saponin approach showed depletion of both host and bacterial DNA to an extent that the bacterial community structure was not retained when looking at 16S rRNA sequencing data. Furthermore, only the Molzym kit showed a satisfying sequencing depth suggesting that although Qiagens kit retained the community structure better than the Saponin approach, the vast amount of host DNA hampered the sequencing effort even after producing amplicons. When employing metagenomic sequencing to evaluate the bacterial:host DNA ratio, the Molzym kit showed up to a 10-fold enrichment of bacterial DNA compared to the Qiagen kit. Selecting an appropriate DNA extraction method is vital when studying epithelial-associated bacteria in gastrointestinal diseases and is essential for unravelling the intricate host-microbiota interactions underlying disease pathogenesis.