An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction
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...
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| Format: | Article |
| Language: | English |
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Elsevier
2016-12-01
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| Series: | Genomics, Proteomics & Bioinformatics |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S1672022916301826 |
| _version_ | 1797332020954660864 |
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| author | Jitendra Kumar Manoj Kumar Shashank Gupta Vasim Ahmed Manu Bhambi Rajesh Pandey Nar Singh Chauhan |
| author_facet | Jitendra Kumar Manoj Kumar Shashank Gupta Vasim Ahmed Manu Bhambi Rajesh Pandey Nar Singh Chauhan |
| author_sort | Jitendra Kumar |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T07:43:38Z |
| format | Article |
| id | doaj.art-118d19379fc74a0bbe3baf517d9eeb43 |
| institution | Directory Open Access Journal |
| issn | 1672-0229 |
| language | English |
| last_indexed | 2024-03-08T07:43:38Z |
| publishDate | 2016-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Genomics, Proteomics & Bioinformatics |
| spelling | doaj.art-118d19379fc74a0bbe3baf517d9eeb432024-02-02T16:42:10ZengElsevierGenomics, Proteomics & Bioinformatics1672-02292016-12-0114637137810.1016/j.gpb.2016.06.002An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA ExtractionJitendra Kumar0Manoj Kumar1Shashank Gupta2Vasim Ahmed3Manu Bhambi4Rajesh Pandey5Nar Singh Chauhan6Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaDepartment of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaDepartment of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaDepartment of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaDepartment of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaAyurgenomics Unit, Translational Research and Innovative Science ThRough Ayurgeomics, Council of Scientific and Industrial Research, Institute of Genomics and Integrative Biology, New Delhi 110020, IndiaDepartment of Biochemistry, Maharshi Dayanand University, Rohtak 124001, IndiaMicrobes 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.http://www.sciencedirect.com/science/article/pii/S1672022916301826Metagenomic DNA extractionGut microbiomeHuman feces16S rRNA |
| spellingShingle | Jitendra Kumar Manoj Kumar Shashank Gupta Vasim Ahmed Manu Bhambi Rajesh Pandey Nar Singh Chauhan An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction Genomics, Proteomics & Bioinformatics Metagenomic DNA extraction Gut microbiome Human feces 16S rRNA |
| title | An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction |
| title_full | An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction |
| title_fullStr | An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction |
| title_full_unstemmed | An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction |
| title_short | An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction |
| title_sort | improved methodology to overcome key issues in human fecal metagenomic dna extraction |
| topic | Metagenomic DNA extraction Gut microbiome Human feces 16S rRNA |
| url | http://www.sciencedirect.com/science/article/pii/S1672022916301826 |
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