An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Abstract

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

The Gut Microbiota and Inflammation: An Overview

“…First, the sample is purified through the use of multiple reagents and centrifugation, allowing for the microbes to be stripped of other components of the faeces. The subsequent step involves lysing bacterial cells by incubating the samples with lysis buffer with agitation, such as vigorous shaking with or without beads, and carrying out further centrifugation [ 12 ]. Following this, the resulting DNA is amplified using approaches such as multiple displacement amplification [ 11 ].…”

“…Firstly, most studies utilise faeces to understand the gut microbiome. Faeces contain many impurities associated with the microbes, affecting the quality of the DNA yielded in the extraction phase, thus affecting interpretation of sample alpha diversity [ 12 ]. Moreover, as the gut contents change throughout the GI tract, the microbial community present in faeces is more suited to the depleted nutrient environment of the latter regions of the GI tract [ 152 ].…”

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

“…found significant differences in metabolite profiles and microbial composition between the top, middle, bottom, and edge positions of stool samples. For this reason, the homogenization of the sample and selection of a representative part of the stool sample are important factors prior to aliquoting and freezing …”

“…For this reason, the homogenization of the sample and selection of a representative part of the stool sample are important factors prior to aliquoting and freezing. [98,100,101] Several methods for fecal pre-preparation exist for storage, including: i) fresh feces freezing at −80°C; ii) centrifuging of fresh feces with or without portions of extracting agent-fecal water; or iii) freeze-drying (fecal powder). [96,97,102,103] The easiest method for fecal storage is homogenization of the fresh stool sample by stirring with a sterile spatula directly in the delivery bag [104][105][106] and then aliquoting a few milligrams to a few grams into feces tubes (i.e., Sarstedt) and storing at −80°C.…”

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

“…Furthermore, from complex matrices, such as those used for most microbial studies, for example soil, stool or swabs, the accuracy of bacterial quantification is affected by the efficiency of the DNA extraction method, which can lead to underestimation of genome abundances if DNA loss occurs [25–27].…”