Microbiomes and the invisible communities within us

microbiology
Microorganisms in our bodies can affect our health in surprising ways, what methods are used to study these effects?
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Since the first human genome sequence was completed in 2000, we have been captivated by the promise of cures for genetic diseases, personalized drugs, and even diets based on our genes. However, studies in microbiology have raised the intriguing prospect of a more complicated picture. This is the idea of a ‘second genome’, perhaps with as much influence in some areas of human health as our ‘primary’ genome, but easier to manipulate and mold for our benefit. The second genome is the microbiome — the collection of all the genes of the microorganisms that live on and in our bodies.
A time to live, a time to die 
Gut feelings
Zoning in on the microbiome with qPCR
Genomics to metagenomics
How to sequence a microbiome
References
A time to live, a time to die
The introduction of antibiotics in the 1930s brought enormous benefits for human health and life expectancy, eliminating many previously fatal diseases, and transforming medical and surgical practices. So it is ironic that the next world-changing discoveries to come from microbiology may focus not on simply killing bacteria, but also on nurturing the correct balance of microorganisms to help maintain our well-being. Microbiologists believe that by occupying environmental niches within our bodies, some microorganisms may displace potentially pathogenic species, or prevent them from ever gaining a foothold. Our microbiota may also help us by producing antimicrobial agents or chemicals that we metabolize. Researchers have noticed the parallels between widespread use of antibiotics and the rise of allergies and autoimmune diseases, suggesting that our native microflora may play a role in strengthening our immune system, which is disrupted by antibiotic use.
 
Gut feelings
Evidence of the importance of a healthy microbiome has come from the impressive successes achieved using fecal transplants from healthy individuals to patients suffering from recurrent Chlostridium difficile (1). Restoring a healthy microflora can be more effective than antibiotics in treating this previously intractable condition. Recognizing the significant benefit to patients, the FDA has recently held a public workshop to discuss the treatment, and has indicated that fecal transplants can be used for some C. difficile infections with the informed consent of the patient (2). Fecal transplants may also benefit other conditions, including colitis (3). Even more surprising are correlations between the make-up of the microbiome and conditions such as autism (4) and atherosclerosis (5). Alterations in the gut microbiota may also explain why obesity is a risk factor for cancer, with changes in gut bacteria shown to be preventative of liver cancer in obese mice (6). These discoveries may lead to therapies aimed at manipulating the microbiome, and certainly confirm the previously unappreciated importance of our second genome.

Zoning in on the microbiome with qPCR
Since disruption in the composition of a microbial community has the potential to explain a variety of disease states, quantitative PCR (qPCR) is an attractive technique for research seeking correlations between the microbiome status and disease. Starting from very small amounts of DNA, qPCR can rapidly screen for specific bacterial species, virulence factor genes, and antibiotic resistance genes, yielding insights into the underlying causes for observed conditions. qPCR takes a snapshot of the true microbial composition in a sample rather than relying on culture of whole organisms. Additionally, qPCR arrays can be designed to target focused panels of microbes and their genes that are known to impact health and disease. QIAGEN has developed a line of Microbial DNA qPCR Arrays and Assays to meet this need, as well as individual assays for specific microbial species and genes.
Genomics to metagenomics
Today, next-generation sequencing (NGS) technologies enable massively parallel sequencing, with the capability of producing millions of sequences simultaneously. These advances, which overcome key limitations of throughput and cost, bring endless new opportunities to explore. NGS has made feasible metagenomic analyses, in which the genetic material, or metagenome, in environmental samples is sequenced and studied. Metagenomics can be used to discover the microorganisms present in a human sample. Identification of species is not dependent on whether or not a particular organism can be cultured in the lab, making it a truer representation of what is really going on in the microbial community and a potential treasure trove of new discoveries. Metagenomics can also zone in on a particular area of interest, for example, assessing the metabolic activity of a sample by analyzing the presence or absence of certain metabolic genes in the metagenome. Analysis of healthy, human microbiomes versus disease microbiomes may shed light on causes, effects, and future therapies for a variety of diseases. Several international consortiums are pooling their resources to catalog the microbiome using the new sequencing technologies. MetaHit, a European research project, has defined 3 ‘enterotypes’ in the world’s population, each characterized by a predominant bacterial population in the gut. The project has also identified bacterial genes that correlate with age and body mass index, indicating potential use as biomarkers (7). Across the water, the NIH-funded Human Microbiome Project is characterizing microorganisms in the nose, mouth, skin, intestine, and urogenital tract.

How to sequence a microbiome
Broadly speaking, microbiome sequencing involves DNA isolation from the sample, followed by one or more preamplification steps, library preparation, and sequencing using an NGS platform. Finally, data analysis enables sensible interpretations and conclusions to be made from the large amount of sequence data generated. 
DNA isolation
This initial step is critical, since downstream processing and amplification procedures cannot compensate for the lack of a good-quality, starting DNA sample. It is important to ensure that the yields of genomic DNA recovered from the sample are as high as possible and that inhibitors present in the sample that would affect later reactions are removed. For isolation of microbial DNA from small volumes of whole blood, swabs, and body fluids including urine, the QIAamp UCP Pathogen Mini Kit provides a rapid protocol (<2 hours for 24 samples). Ultraclean production (UCP) practices provide the added reassurance that the kit components are delivered free of contaminating microbial DNA. For isolation of microbial DNA from stool, the QIAamp DNA Stool Mini Kit removes the high amounts of PCR inhibitors present in this sample type. Purification is fast and yields are high (typically 10–30 µg from 220 mg stool). Removal of human DNA from samples is a desirable requirement for microbiome studies, as downstream sequencing of human DNA takes up sequencer capacity, and human sequence information will need to be removed during data analysis. QIAGEN R&D scientists are currently developing products for the targeted removal of human DNA. 
Next-generation sequencing
QIAGEN’s portfolio of dedicated products for NGS allows generation of high-quality, target-enriched samples and libraries that will deliver the most accurate data possible. The portfolio is fully compatible with Illumina MiSeq and HiSeq sequencing platforms, as well as Ion Torrent instruments from Life Technologies.

References
  1. Els van Nood, M.D. et al. (2013) Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile. N. Engl. J. Med. 368, 407.
  2. FDA (May 2013) Public Workshop: Fecal Microbiota for Transplantation. www.fda.gov/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/ucm341643.htm
  3. Kunde, S. et al. (2013) Safety, tolerability, and clinical response after fecal transplantation in children and young adults with ulcerative colitis. J. Pediatr. Gastroenterol. Nutr. 56, 597.
  4. Mulle, J.G., Sharp, W.G., and Cubells, J.F. (2013) The gut microbiome: a new frontier in autism research. Curr. Psychiatry Rep. 15, 337.
  5. Koeth, R. A. et al. (2013) Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature Medicine 19, 576.
  6. Yoshimoto, S. et al. (2013) Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature doi:10.1038/nature12347.
  7. Arumugam, M. et al. (2011) Enterotypes of the human gut microbiome. Nature 473, 174.

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QIAseq Library Quant Array Kit
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GeneRead Size Selection Kit (50)
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