title: 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 (>23kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8±3.8ng/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.
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India.
authors.forename: Jitendra
authors.initials: J
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India.
authors.forename: Manoj
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India.
authors.forename: Shashank
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India.
authors.forename: Vasim
authors.initials: V
authors.lastname: Ahmed
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India.
authors.forename: Manu
authors.initials: M
authors.lastname: Bhambi
authors.affiliation: Ayurgenomics Unit, Translational Research and Innovative Science ThRough Ayurgeomics, Council of Scientific and Industrial Research, Institute of Genomics and Integrative Biology, New Delhi 110020, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Pandey
authors.affiliation: Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, India. Electronic address: nschauhan@mdurohtak.ac.in.
authors.forename: Nar Singh
authors.initials: NS
authors.lastname: Chauhan
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D056186:Metagenomics / Q000379:methods*; D064307:Microbiota; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D012336:RNA, Ribosomal, 16S; D004247:DNA
keywords: 16S rRNA; Gut microbiome; Human feces; Metagenomic DNA extraction