Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Nechvatal, Jordan M.; Ram, Jeffrey L.; Basson, Marc D.; Namprachan, Phanramphoei; Niec, Stephanie R.; Badsha, Kawsar Z; Matherly, Larry H.; Majumdar, Adhip P.N.; Kato, Ikuko

doi:10.1016/j.mimet.2007.11.007

Cited by 154 publications

(147 citation statements)

References 18 publications

Supporting

Mentioning

139

Contrasting

Unclassified

Order By: Relevance

“…A number of efforts to optimize a methodology for metagenomic DNA isolation from feces have been undertaken [11], [13], [15], [20]. Although progress has been made in this regard, problems of limited applicability ( e.g.…”

Section: Resultsmentioning

confidence: 99%

“…Although progress has been made in this regard, problems of limited applicability ( e.g. , microbial diversity studies only) and acceptability due to their complex process, poor metagenomic DNA quality, host genomic DNA contamination, low yield, and high cost have still left scope for a methodology to overcome the shortcomings [11], [17], [18], [19], [20], [21].…”

Section: Resultsmentioning

confidence: 99%

“…In summary, current methodology has been successfully used to isolate the metagenomic DNA from one-month-old frozen fecal sample stored at −86 °C with a minimal effect on yield and quality, which was a challenge as per reported metagenomic DNA isolation studies [13], [14], [20], [21], [22].…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, human genomic DNA remnants in metagenomic DNA affect the metagenomic sequence data output, thereby increasing per base sequencing cost [6]. To overcome these challenges, many metagenomic DNA extraction protocols are being standardized, which include phenol/chloroform enzymatic lysis and freeze thaw [11], [16], [17], [18], [19], [20], [21], [22]. These kits have improved the quality of metagenomic DNA.…”

Section: Introductionmentioning

confidence: 99%

“…These kits have improved the quality of metagenomic DNA. However, great concerns remain pertaining to microbial diversity biasness and human genomic DNA contamination [12], [17], [20]. The cost per sample, amount of sample, and associated impurities are other issues which may be improved upon.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Kumar

Gupta

et al. 2016

Genomics, Proteomics &Amp; Bioinformatics

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Kumar

Gupta

et al. 2016

Genomics, Proteomics &Amp; Bioinformatics

View full text Add to dashboard Cite

show abstract

Getting Started with Microbiome Analysis: Sample Acquisition to Bioinformatics

et al. 2014

View full text Add to dashboard Cite

Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture‐independent methods to study microbiota using high‐throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators. Curr. Protoc. Hum. Genet. 82:18.8.1‐18.8.29. © 2014 by John Wiley & Sons, Inc.

show abstract

Fecal bacterial diversity of human‐habituated wild chimpanzees (Pan troglodytes schweinfurthii) at Mahale Mountains National Park, Western Tanzania

Szekely

Singh

Marsh

et al. 2010

American J Primatol

View full text Add to dashboard Cite

Although the intestinal flora of chimpanzees has not been studied, insight into this dynamic environment can be obtained through studies on their feces. We analyzed fecal samples from human-habituated, wild chimpanzees at Mahale Mountains National Park, Tanzania, and compared microbial community profiles to determine if members of the same social group were similar. Between July and December 2007, we collected fresh fecal samples from 12 individuals: four juveniles, four adolescents, and four adults, including three parent-offspring pairs. Each sample was analyzed using Terminal-Restriction Fragment Length Polymorphism of amplified 16S rRNA genes. Twelve different profiles were generated, having between 1 and 15 Terminal-Restriction Fragments (T-RFs). Overall, a total of 23 different T-RFs were produced. Putative assignments of T-RFs corresponded to the phyla Firmicutes (Clostridia, Bacilli, and Lactobacilli), Bacteroidetes, Tenericutes (Mollicutes Class), Actinobacteria, and Proteobacteria, as well as to uncultured or unidentified organisms. Firmicutes and Bacteroidetes phyla and Mollicutes Class were the most commonly assigned in 11, 8, and 8 of the samples, respectively, with this being the first report of Mollicutes in wild chimpanzees. Principal Components Analysis (PCA) revealed clustering of nine samples, and 80.5% of the diversity was accounted for by three samples. Morisita indices of community similarity ranged between 0.00 and 0.89, with dissimiliarity (<0.5) between most samples when compared two at a time. Our findings suggest that, although phylotypes are common among individuals, profiles among members of the same social group are host-specific. We conclude that factors other than social group, such as kinship and age, may influence fecal bacterial profiles of wild chimpanzees, and recommend that additional studies be conducted.

show abstract

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Cited by 154 publications

References 18 publications

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

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

Getting Started with Microbiome Analysis: Sample Acquisition to Bioinformatics

Fecal bacterial diversity of human‐habituated wild chimpanzees (Pan troglodytes schweinfurthii) at Mahale Mountains National Park, Western Tanzania

Contact Info

Product

Resources

About