You and Your Microbiome – Part 2

  • Global Obesity Epidemic is Linked to Gut Microbiome
  • DNA Sequence-Based Microbiomes Accurately Associate with Obesity
  • Blue Agave Margaritas Contain Beneficial Gut Microbes
  • Investments in Microbiome-based Therapies on the Rise, but is there Hype?

Last August, my post entitled Meet Your Microbiome: The Other Part of You dealt with growing recognition that trillions of microbes—mostly bacteria but also fungus—reside in and on each of us, and influence our health status. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we contact—family, pets, and close friends.

Since then, I’ve collected a string of microbiome articles delving into the implications of this dynamic, symbiotic relationship, and selected some topics that I thought were “blogworthy.” This Part 2, as it were, focuses on overweight/obesity, microbiome therapy, and burgeoning business opportunities.

Continue reading

Meet Your Microbiome: The Other Part of You

  • Like it or not, and for better or worse, next-generation sequencing is revealing that you and your bacterial microbiome have an inextricable biological relationship.
  • “RePOOPulating” the gut:  a clinical study of “synthetic stool” as a better alternative to fecal transplant.
  • Microbiome movies, fungus too, and much more.

What’s in your microbiome? Why does it matter?

Writing this blog was inspired by reading Michael Pollan’s recent article in The NY Times Magazine entitled “Some of my best friends are bacteria,” which is an engaging story about Michael having his microbiome sequenced as part of the American Gut project. He notes at the beginning that each of us has several hundred microbial species with whom we share our body, and that these bacteria—numbering ~100 trillion—are living (and dying) right now on the surface of our skin, mouth, and intestine “where the largest contingent of them will be found, a pound or two of microbes together forming a vast, largely uncharted interior wilderness that scientists are just beginning to map.” The sheer numbers of these microbes, he adds, makes us only ~10% human—for every human cell there are ~10 resident microbes—most being “harmless freeloaders” or “favor traders” (i.e. symbiotic), and only a tiny number of pathogens. Furthermore, “[t]his humbling new way of thinking about the self has large implications for human and microbial health, which turn out to be inextricably linked.”

americangut

If you want to know what’s in your gut, you can participate in this world wide study by registering at www.americangut.org. According to the website, you’ll be asked to ‘fill out a diet & lifestyle questionnaire (online) with such things as age, gender, weight, have you taken antibiotics lately, any conditions we should know about it, and so on.’ Anyone over the age of 3 months can participate in the study, and you will have the opportunity to provide a stool, tongue and/or palm sample via an at-home sample kit.

Microbes and Your Health

As more microbiome data is obtained from the American Gut project and analogous studies, correlations with each person’s health status can be made. Having the “wrong” kind of microbes may be associated with predisposition to obesity or certain chronic diseases, for example. Such information may then be used to prescribe dietary or other sources supplemental probiotics. An extreme outcome could involve “fecal transplants” (i.e., fecal microbiota transplantation or fecal bacteriotherapy) wherein a healthy person’s fecal microbiota are installed into a sick person’s gut.

My PubMed searches of these terms led to a number of publications, such as that by Christopher. R. Kelly et al. at Women and Infant’s Hospital, Brown University Alpert School of Medicine, who reported promising results for relapsing Clostridium difficile infection in a small study of 26 patients. Coincidentally, on June 18th there was a report that the US Food & Drug Administration (FDA) is “dropping plans to tightly control” fecal transplants that are “becoming increasingly popular for treating people stricken by life-threatening infections of the digestive system.” This report went on to say that, in the FDA’s view, “such a treatment should only be given to patients who have exhausted other treatment options and who have given consent and been informed that it is an experimental procedure with risks.”

RePOOPulating the gut:  a better alternative?

Among the concerns for fecal transplants are pathogen transmission, patient acceptance and inability to standardize the treatment regime, states Elaine O. Petrof and her colleagues in a study entitled Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut, recently published in Microbiome (2013). This online, open-access article is well worth a quick read if you’re interested in the experimental details, which take advantage of next-generation sequencing as a key method for quite sophisticated identification and analysis of microbes. In brief, a stool substitute preparation—made from 33 purified intestinal bacterial cultures derived from a single healthy donor—was used to treat two patients who had failed at least three courses of metronidazole or vancomycin. Pre-treatment and post-treatment stool samples were analyzed by 16S rRNA sequencing using the Ion Torrent platform. Both patients were infected with a hyper virulent C. difficile strain, but following treatment each reverted to their normal bowel pattern within 2-3 days and remained symptom-free at 6 months. Analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples, but constituted >25% of the sequences 6 months after treatment.

gutlining

Bacteria on the gut lining. Image: Cardiff University

Microbiome Movies

Relatively low-cost deep-sequencing technology has enabled novel studies aimed at obtaining, in effect,  “moving pictures of the human microbiome,” which is the attention-grabbing title of a study by J. Gregory Caparaso et al. in Genome Biology. This landmark publication in 2011 presented the largest human microbiota time-series analysis to date, covering two individuals each at 4 body sites over 396 time-points. One male and one female each provided gut (feces), mouth, left palm, and right palm samples over 15-mo and 6-mo periods, respectively, Variable regions of 16S ribosomal RNA (rRNA) in each sample were amplified by PCR and sequenced on an Illumina instrument. The following stated results and conclusions may surprise you, as they did me:

“We find that despite stable differences between body sites and individuals, there is pronounced variability in an individual’s microbiota across months, weeks and even days. Additionally, only a small fraction of the total taxa found within a single body site appear to be present across all time points, suggesting that no core temporal microbiome exists at high abundance (although some microbes may be present but drop below the detection threshold). Many more taxa appear to be persistent but non-permanent community members.”

“Because of the immense subject-to-subject variability in the microbiome, studies examining temporal variability, which give a view of dynamics beyond the static pictures previously available, have the potential to transform our understanding of what is ‘normal’ in the human body, and, perhaps, to develop predictive models for the effects of clinical interventions.”

If you’re questioning whether the above study of only two individuals—albeit for many time points—reflects a wider population, rest assured that it apparently does. The recently completed 5-yr Human Microbiome Project (HMP) launched in 2008 involved 242 volunteers, more than 5,000 samples were collected from tissues from 15 (men) and 18 (women) at body sites such as mouth, nose, skin, lower intestine (stool) and vagina. According to the HMP wiki site, this project’s discoveries include:

  • Microbes contribute more genes responsible for human survival than humans’ own genes. It is estimated that bacterial protein-coding genes are 360 times more abundant than human genes.
  • Microbial metabolic activities—for example, digestion of fats—are not always provided by the same bacterial species. The presence of the activities seems to matter more.
  • Components of the human microbiome change over time, affected by a patient disease state and medication. However, the microbiome eventually returns to a state of equilibrium, even though the composition of bacterial types has changed.

Consequently, defining what is “normal” and whether a “core” community of microbes exists is quite complicated, and will likely remain a topic of great interest and debate. In this regard, I found an overview by Dirk Gevers et al. well worth reading. Here’s one section of text that reiterates what I think are important points, and elaborates upon the above second bullet point about microbial metabolic activities, i.e. functional core:

“A potentially more universal ‘core’ human microbiome emerged during the consideration of microbial genes and pathways carried throughout communities’ metagenomes. While microbial organisms varied among subjects as described above, metabolic pathways necessary for human-associated microbial life were consistently present, forming a functional ‘core’ to the microbiome at all body sites. Although the pathways and processes of this core were consistent, the particular genes that implemented them again varied. Microbial sugar utilization, for example, was enriched for metabolism of simple sugars in the oral cavity, complex carbohydrates in the gut, and glycogen/peptidoglycan degradation in the vaginal microbiome. The healthy microbiome may thus achieve a consistent balance of function and metabolism that is maintained in health, but with fine-grained details personalized by genetics, early life events, environmental factors such as diet, and a lifetime of pharmaceutical and immunological exposures.”

Lest you think that such data are collections of facts devoid of utility, think again after reading the following excerpts from the abstract of a publication by Fredrik H. Karlsson et al. in Nature 2013 entitled Gut metagenome in European women with normal, impaired and diabetic glucose control.

“Type 2 diabetes (T2D) is a result of complex gene–environment interactions, and several risk factors have been identified, including age, family history, diet, sedentary lifestyle and obesity. Statistical models that combine known risk factors for T2D can partly identify individuals at high risk of developing the disease. However, these studies have so far indicated that human genetics contributes little to the models, whereas socio-demographic and environmental factors have greater influence.…Here we use shotgun sequencing to characterize the fecal metagenome of 145 European women with normal, impaired or diabetic glucose control. We observe compositional and functional alterations in the metagenomes of women with T2D, and develop a mathematical model based on metagenomic profiles that identified T2D with high accuracy.”

Microbiomes in Homes and Hospitals

Mapping the great indoors—an engaging NY Times article by Peter A. Smith—tells about microbiologists who are using sequencing to “take a census” of what lives in our homes with us and how we “colonize” spaces with other species — viruses, bacteria, microbes. One group has sampled the “microbial wildlife” in 1,400 homes across the USA in a project called The Wild Life of Our Home, which relies on volunteers to swab pillowcases, cutting boards and doorjambs, then send samples in for analysis. Although data are still being analyzed, an earlier study of 40 homes in 9 locations around the Raleigh-Durham area of North Carolina has been published by Robert R. Dunn et al. in PLoS ONE entitled Home Life: Factors Structuring the Bacterial Diversity Found within and between Homes. Among their findings were the following.

“[E]ach of the sampled locations harbored bacterial communities that were distinct from one another” [and that] “the presence of dogs had a significant effect on bacterial community composition in multiple locations within homes as the homes occupied by dogs harbored more diverse communities and higher relative abundances of dog-associated bacterial taxa. Furthermore, we found a significant correlation between the types of bacteria deposited on surfaces outside the home and those found inside the home, highlighting that microbes from outside the home can have a direct effect on the microbial communities living on surfaces within our homes.”

Your dirty dog? (Bing Images)

Your dirty dog? (Bing Images)

Some of you might be wondering what microbes your dog is depositing in your home, while others might be asking about cats. I haven’t a clue about your dog, but cats weren’t directly assessed in this study. Thirteen of the houses had only dogs as pets and the influence of dogs is likely to be greater than cats because of their large size and need to go outdoors. Only three houses had cats but not dogs (three houses had both), and the investigators judged this to be too small a sample size for cats.

The figure shown below taken from the aforementioned Dunn et al. publication tracks 9 sites sampled with 5 sources of microbes. The authors note that “[t]hese results show changes in the relative importance of individual sources across sites, not comparisons across sources within sites. For example, these results show that soil is a more important source of bacteria on door trims than on cutting boards, but these results cannot be used to directly compare the relative importance of soil versus leaves as sources of bacteria at individual locations.” By the same token, the results show that human skin is a more important source of bacteria on toilet seats than on cutting boards.

bacteriaproportion

Source tracking analysis showing relative proportion of bacteria at each sampling site associated with given sources. Values represent median percentages. Warmer colors indicate greater influences of particular sources across the sites (Robert R. Dunn et al. PLoS ONE 2013).

Microbial fingerprints

Roughly 1.7 million hospital-associated infections are reported each year in the USA, and the pathogens that cause them must come from somewhere. Beth Mole (too bad she wasn’t named Millie!) in Nature-News 2013 notes that patients leave a microbial mark on hospitals. This attention getting punch line refers to preliminary findings from the Hospital Microbiome Project that, according to its website, “aims to collect microbial samples from surfaces, air, staff, and patients from the University of Chicago’s new hospital pavilion in order to better understand the factors that influence bacterial population development in healthcare environments.”

According to Beth Mole, “…even in areas with long-term inhabitants, [Jack] Gilbert’s team has found no lingering pathogens. ‘Over the first four months of observations, we’ve seen nothing that concerns us,’ he says.” However, “Gilbert and his team found significant differences between microbial communities in individual hospital rooms. Patients who stayed for only short periods, such as those undergoing elective surgery, had a transient influence on their rooms’ microbial communities; after cleaning, the rooms reverted to a pre-patient state.” In contrast, and of concern, ‘[m]icrobes from long-term patients—including people with cancer or those who had received organ transplants—had time to settle into the rooms. The patients’ microbial fingerprints lingered after they checked out of the hospital, even after their rooms were cleaned.” But even in areas with long-term inhabitants, Gilbert’s team has found no lingering pathogens. “Over the first four months of observations, we’ve seen nothing that concerns us,” he says.”

Thinking Big: The Global Microbiome

If you now muse about greatly expanding the scope of the aforementioned studies to a global scale, you’d be thinking about something that has already been proposed. The Earth Microbiome Project is a “massively multidisciplinary effort to analyze microbial communities across the globe” that proposes to “characterize the Earth by environmental parameter space into different biomes and then explore these using samples currently available from researchers across the globe.” The project intends to “analyze 200,000 samples from these communities using metagenomics, metatranscriptomics and amplicon sequencing to produce a global Gene Atlas describing protein space, environmental metabolic models for each biome, approximately 500,000 reconstructed microbial genomes, a global metabolic model, and a data-analysis portal for visualization of all information.” We’ll all have to stay tuned on this rather ambitious project to learn what is found and, more importantly, what are the conclusions—and whether time-dependent variability is accounted for.

By the way, if you’re wondering about the total global microbiome, Addy Pross’ book entitled What is Life? How Chemistry Becomes Biology states that the earth’s bacterial biomass is estimated to be 2 × 1014 tons, which is sufficient to cover the earth’s land surface to a depth of 1.5 meters!

Microbiome-mania & Toilet-phobia

There seems to be a surge in extending microbiome analysis to many other contexts. Some of these findings are surprising—and prompting jokes—or might scare you about microorganisms present in various places or on things that we come into contact with, whether we know it or not.

Initial, a UK-based provider of hygiene services to businesses and organizations, announced in an April 2013 press release, its research that “…lifts the lid on the grimy state of Britain’s office kitchens.” Being a provider of cleaning services, Initial was presumably quite pleased to inform the public about the following findings.

“Swab testing of a sample of communal workplace kitchens showed that 75% of work surfaces were home to more bacteria than an average feminine sanitary bin. Half also harboured dangerously high levels of coliforms, bacteria present in feces, which can lead to outbreaks of gastrointestinal disease. Over a quarter of the draining boards tested registered more than four times the level of coliforms considered to be safe.”

“The handles of shared fridge-freezers were also shown to be bacteria-rife, with a third carrying high levels of coliforms, whilst 30% of shared microwaves were also shown to be contaminated around the handles and buttons.”

“Tea drinkers were no more hygienic, with over 40% of kettle handles found to be carrying high levels of bacteria, and significantly exceeding the bacteria levels on toilet doors. Also, tested were cupboard, dishwasher and waste bin handles, with the cleanest appliance in the kitchen proving to be the water cooler.”

Speaking of toilets, they seem to be in vogue in metabolome studies. For example, Emma Innes reported online that women’s handbags contain more bacteria than the average toilet seat. The dirtiest item in an average handbag is hand cream—it carries more bacteria than the average toilet seat. Leather handbags carry the most bacteria because the spongy texture provides “perfect growing conditions.” An online article by Harold Maass noted that British researchers found that the average barbecue grill in the UK has more than twice as many germs as the typical toilet seat. Maybe someone has already invented a disposable barbeque grill cover similar to what’s used to cover toilet seats, but obviously nonflammable.

Sanitor Mfg Co. began producing toilet seat covers and dispensers in the USA in 1931.  Seems they are working as your handbag and your barbeque may both harbor more germs than a toilet seat.

Sanitor Mfg Co. began producing toilet seat covers and dispensers in the USA in 1931. Seems they are working as your handbag and your barbeque may both harbor more germs than a toilet seat.

Although humorously entitled as Lifting the lid on toilet plume aerosol, this review article recently published the American Journal of Infection Control examines the evidence regarding toilet plume bioaerosol generation and infectious disease transmission. Here’s a quote of the results of this review of existing literature. “The studies demonstrate that potentially infectious aerosols may be produced in substantial quantities during flushing. Aerosolization can continue through multiple flushes to expose subsequent toilet users. Some of the aerosols desiccate to become droplet nuclei and remain adrift in the air currents. However, no studies have yet clearly demonstrated or refuted toilet plume-related disease transmission, and the significance of the risk remains largely uncharacterized.” Like many investigations, the stated conclusions call for more data: “[a]dditional research in multiple areas is warranted to assess the risks posed by toilet plume, especially within health care facilities.

By the way, the internet has various references to a 6-foot diameter (dare I say) zone wherein toilet aerosol may spread, so keep your toothbrush at a safe distance or consider purchasing a toothbrush sanitizer—of which there are many—after doing your homework on which devices have been validated, and against what, as I did by searching PubMed for “toothbrush sanitizer” etc.

Mycobiome:   Fungus In, On and Among Us

Oh…let’s not forget about fungus—a member of a large group of microorganisms that includes yeasts and molds, as well as mushrooms. These organisms are classified as a kingdom, Fungi, and the discipline of biology devoted to the study of fungi is known as mycology. My PubMed search of “mycobiome” gave only 10 hits, which is far less than ~4,000 found for “microbiome.” The earliest mycobiome publication was entitled “Characterization of the oral fungal microbiome (mycobiome) in healthy individuals” by Mahmoud A. Ghannoum et al. (PLoS Pathogens 2010). This study—which used pyrosequencing to characterize fungi present in the oral cavity of 20 healthy individuals—revealed the “basal” oral mycobiome profile of the enrolled individuals and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. The oral mycobiome of at least 20% of the enrolled individuals included the four most common pathogenic fungi—Candida (present in 75% of the cohort; mostly C. albicans), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). The authors said that “[i]t is possible that the pathogenicity of these fungi is controlled in healthy individuals by other fungi in the oral mycobiome, as well as a functional immune system.”

Candida albicans (Wikipedia via Bing Images)

Candida albicans (Wikipedia via Bing Images)

More recently, Heidi H. Kong and coworkers in Nature 2013 published a report entitled Topographic diversity of fungal and bacterial communities in human skin. This study involved 10 healthy individuals and used sequencing to analyze fungal and bacterial communities sampled from 14 skin sites that included face, chest, arms, ears, nostrils, head, and feet. Some salient points taken from the abstract are as follows.

“Eleven core-body and arm sites were dominated by fungi of the genus Malassezia, with only species-level classifications revealing fungal-community composition differences between sites. By contrast, three foot sites—plantar heel, toenail and toe web—showed high fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that physiologic attributes and topography of skin differentially shape these two microbial communities. These results provide a framework for future investigation of the contribution of interactions between pathogenic and commensal fungal and bacterial communities to the maintenance of human health and to disease pathogenesis.”

Lastly, Who was First?

Who was “first-to-publish” and what was envisaged have always been of interest to me. In researching this posting, I became curious about who introduced the term “microbiome” to generally describe concepts of the type represented in the various aforementioned articles. It’s not easy to be find or establish “first-ever” publications, so I took the easy way out by doing a PubMed search wherein “microbiome” is in the title and/or abstract. Of the ~1,400 articles found, the earliest was entitled New technologies, human-microbe interactions, and the search for previously unrecognized pathogens by David A. Relman at Stanford University, and appeared in Journal of Infectious Diseases in 2002. The following conclusion from his abstract is quite prescient, and I’m amazed by how far and fast microbiome science has progressed since then.

“The development and clinical application of molecular methods have led to the discovery of novel members of the endogenous normal flora as well as putative disease agents. Current challenges include the establishment of criteria for disease causation and further characterization of the human microbiome during states of health. These challenges and the goal of understanding microbial contributions to inflammatory disease may be addressed effectively through the thoughtful integration of modern technologies and clinical insight.”

As always, your comments are welcome—especially if you know of a particularly interesting “microbiome” report that you’d like to share with me and other readers of this blog.

Postscript

After writing this blog, GenomeWeb reported on August 8th that The Wellcome Trust has awarded $2 million to fund Lindsay Hall, a researcher at the University of East Anglia and the Institute of Food Research, who will seek to find out how bacteria that are beneficial to humans help protect against diseases in the early phases of life, using high-throughput sequencing tools to find out more about the microbial communities that colonize the human body soon after birth. “We are planning to use 16S rRNA-based microbiota community analysis, metagenome, and whole genome sequencing to define and characterize early-life microbiota samples,” Hall told GenomeWeb Daily News in an e-mail. The earliest parts of human life are a critical period in terms of the microbiome because at birth the human gut is completely bacteria-free, Hall explained, noting that the processes that follow birth and lead to microbial colonization are not fully understood. Having a better understanding of these processes could lead to treatments for diseases such as bacterial gastroenteritis, she said. This infectious disease is an increasing cause of infant death in the developing world, and the treatment involves antibiotics, but resistance to antibiotics is increasing and antibiotics also may reduce natural defenses against infection. Under this project, she will look to understand how antibiotics can disrupt these microbial communities, and search for probiotic bacteria.

Three Takeaways from the 3rd Next-Generation Sequencing Conference

  • Exciting potential of direct sequencing of modified DNA 
  • Small holes with big promise but bigger challenges 
  • Paleogenomics:  sequencing ancient DNA—how old can you go? 

Sometimes small scientific meetings have big impacts on one’s impressions, which was certainly my experience at the 3rd Next-Generation Sequencing (NGS) conference in San Francisco on June 19-21, 2013. Of the many interesting presentations (click here for all speakers and abstracts), three completely different topics struck me the most: Pacific Biosystems’ uniquely powerful single-molecule real-time (SMRT) sequencing of modified DNA, Sequencing-pioneer Prof. David Deamer’s update on Nanopore’s advances and challenges, and the new field of Paleogenomics involving sequencing old DNA. With apologies to all of the other speakers, and admitting personally biased selection, here are my comments about these three topics.

Pacific Biosystems: direct sequencing of modified DNA

jonas

Dr. Jonas Korlach co-invented SMRT technology with Stephen Turner, Ph.D., PacBio Founder and Chief Technology Officer, when the two were graduate students at Cornell University. Dr. Korlach joined PacBio as the company’s eighth employee in 2004. Dr. Korlach was appointed Chief Scientific Officer at PacBio in July, 2012.

Pacific Biosystems (PacBio) deserves a lot of credit for being able to overcome numerous technical challenges facing commercialization of its SMRT sequencing system, which offers some uniquely powerful capabilities. (I’ll save a bit of time and space by refraining from describing how this complex system works, but I encourage you to take advantage of various videos and other technical information available at PacBio’s website.) In addition to providing amazingly long read lengths (up to 20kb) to facilitate genome assembly, SMRT sequencing gives data related to kinetics of nucleotide incorporation. Algorithms for differentiating rate of incorporation of A, G, C or T opposite a cognate nucleotide position in the template strand for various sequence contexts within the “footprint” of a DNA polymerase can also differentiate modified template positions. In other words, the average rate of incorporation of G opposite C is different than that opposite 5-methylcytosine (5-mC). This difference in kinetics allows direct determination of epigenetic methylation patterns in DNA, which was the focus of an excellent presentation by PacBio CSO Jonas Korlach. Direct epigenetic sequencing of 5-mC is completely novel and offers a significant advantage by obviating the need to carrying out so-called ‘bisulfite conversion chemistry’ prior to sequencing. Commercial kits are available for bisulfite conversion but require extra time, can be very tricky, and utilize more sample than may be available—especially for limited amounts of clinical biopsies.

I subsequently checked PacBio’s website and found a white paper pdf stating that unique kinetic characteristics have been observed for over 25 types of base modifications, such as those shown below and for these reasons:

Molecular structures and abbreviations for modified bases directly identifiable by SMRT sequencing (taken from PacBio white paper).

Molecular structures and abbreviations for modified bases directly identifiable by SMRT sequencing (taken from PacBio white paper).

Especially exciting to me was Dr. Korlach’s brief mention at the end of his talk that SMRT could be used for direct sequencing of phosphorothioate (PS) linkages in DNA. While “man-made” PS modifications in synthetic DNA are well known, naturally occurring PS-DNA is a relatively recent—and quite surprising—discovery still being elucidated. A 2013 review (click here for pdf) of this novel and fascinating type of naturally occurring modified DNA states that “physiological PS modification is widespread in bacteria and occurs in diverse sequence contexts and frequencies [approximately 300 – 3,000 PS per 106 nucleotides] in different bacterial genomes, implying a significant impact on bacteria.” Bacterial PS-DNA has been shown to be introduced by a post-replicative biochemical pathway associated with a cluster of five genes, and is implicated in site-specific restriction and, more recently, chemical reducing capacity to protect bacteria against peroxide. PS linkages in DNA can have SP or RP stereochemistry at the phosphorus as shown below; however, all bacterial PS-DNA examined to date occurs in the RP form.

Generalized molecular structure of SP and RP PS-DNA linkages (taken from RS Phosphorothioates Wikipedia).

Generalized molecular structure of SP and RP PS-DNA linkages (taken from RS Phosphorothioates Wikipedia).

I later contacted Dr. Korlach to get more information about PS sequencing by SMRT and he referred me this video (~17 minutes) and conference abstract. In response to my question about whether SMRT sequencing could differentiate SP from RP stereochemistry, he replied that he and his collaborators have looked at this possibility but he couldn’t comment at this time because the work was ongoing and would be published in the future.

While awaiting publication of those findings, it’s interesting—I think—to speculate about other applications of SMRT direct sequencing of modified DNA. One intriguing possibility is determining the extent of, and genomic loci for, 5-fluoro-2′-deoxyuridine incorporation into DNA that heretofore has only been studied using indirect methods to decipher mechanisms of action of various 5-fluoropyrimidine anticancer agents.

What other possible applications of SMRT direct sequencing of modified DNA can you suggest?  (Please include in the comments section.)

Nanopore sequencing:  small holes with big promise but bigger challenges

When I presented a Church, Deamer, Branton et al. patent that broadly describes nanopore sequencing of DNA (see below) to my former marketing colleagues at Applied Biosystems Inc. (ABI) in 1998, they enthusiastically asked “how soon can we sell a nanopore sequencer?” After I told them the patent was prophetic and had no actual data, they disappointedly said “too bad, let us know when it’s ready.” Well, it’s now 15 years later, and many folks like me are still waiting for that commercialization date, despite hundreds of publications on many different variations of the basic concept.

Consequently, I attended Prof. David Deamer’s presentation with the hope of learning when some type of nanopore sequencer would finally be introduced by any one of several companies in this space—notably Oxford Nanopore Technologies (ONT), whose stellar Technology Advisory Board includes Prof. Deamer.

david

David W. Deamer is a Research Professor in the Department Chemistry & Biochemistry at UC Santa Cruz where his primary research area concerns the manner in which linear macromolecules traverse nanoscopic channels.

Prof. Deamer presented an excellent update starting with a stylized version of his original lab notebook sketch of the technology (see below). He then discusses some of the incremental progress—and many remaining challenges—for nanopore sequencing (check out reviews by Dunbar et al and others by searching “nanopore sequencing” on PubMed). He concluded with a description of recent results obtained by a group led by Prof. Mark Akeson, his long-time colleague and collaborator at UCSC. Among various innovations, a processive DNA polymerase is used to control translocation by ratcheting. Although the sequencing results presented were limited to only ~10 bases in a model oligonucleotide, a well-known and rather critical attendee—who I’ll keep anonymous—said during Q&A that “these were the most promising data I’ve seen so far.” That attendee then asked about ONT’s timeline for commercialization, to which Prof. Deamer said “he doesn’t speak for the company, but thinks that something might be introduced in another 6 months or so.”

nanopore

Deptiction of nanopore sequencing method described in Church, Deamer, Branton et al. patent US 5,795,782.

At the risk of sounding like a pessimist, but based on my past experience where timelines for developing complex automated systems always took much longer than desired, I’d be very surprised if that “something” is launched by the end of 2013. Hopefully I am wrong so I’ll be on the look out just in case.

In the meantime, while we all await such an event, you can read about several thought-provoking nanopore sequencing-related topics:

Paleogenomics:  sequencing ancient DNA—how old can you go?

Relative to evolutionary time-spans, the study of paleogenetics is not old—going back to 1963 and Linus Pauling; however, very, very old (aka “ancient”) DNA is now “sequenceable” using modern NGS technologies. Just how old is “ancient” and what is the projected age-limit for sequenceable DNA were two questions I had in mind at the outset of the presentation by Prof. Eske Willerslev, who has been a pioneer in this field.

eske

Prof. Eske Willerslev is a Danish evolutionary biologist at Copenhagen University and leader of the Ancient DNA and Evolution Group. He has received the Genius Award (Geniusprisen) of Danish Science journalists for his combination of groundbreaking research with an aggressive media strategy. Before becoming a scientist he lived for several years as a trapper in Siberia with his twin brother, anthropologist Rane Willerslev.

The presentation by Prof. Willerslev was rapidly delivered and jam-packed with snippets of results from numerous studies, which is another way of saying here that it was impossible for me to take notes from which to reconstruct a synopsis ex post facto using cited publications. On the other hand, I did get the following answers to my two probing questions.

Just how old is ‘ancient’ DNA?  

Prof. Willersley said that a draft genome from a ~700 thousand years before present (~700k yr BP) horse bone found at Thistle Creek, Canada represents the oldest full genome sequence determined so far, and by almost an order of magnitude. This stunning—to me—achievement, which was published in Nature online several days after the conference and has received considerable attention because of the significance of its findings with regard to “recalibrating Equus evolution.” As stated in the abstract of this publication, “[f]or comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr BP), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski’s horse (E. f. przewalskii) [pictured below] and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr BP), twice the conventionally accepted time to the most recent common ancestor of the genus Equus.”

horse

Przewalski’s horse at Khustain Nuruu National Park in Mongolia. These horses are smallish and stocky in comparison to domesticated horses, with shorter legs that are often faintly striped, typical of primitive markings. The Przewalski’s horse has 66 chromosomes, compared to 64 in all other horse species. All Przewalski horses in the world are descended from 9 of the 31 horses in captivity in 1945. These 9 horses were mostly descended from ~15 captured around 1900. The total number of these horses by the early 1990s was over 1,500.

Interestingly, the aforementioned Nature publication reports using a combination of Illumina and Helicos sequencing, with the latter’s single-molecule sequencing capabilities providing an “advantageous complement” to the former’s data, as previously described. Since Helicos is now defunct, it will be interesting to see if such methodological complementarity can instead be provided by PacBio’s single-molecule sequencing.

How old can you go?  

As for “how old can you go” and still get sequenceable DNA, Prof. Willerslev said at the conference that “1 or 2 million years old should be possible.” A subsequent article by Millar & Lambert in Nature News & Views entitled Towards a million-year-old genome confirmed this and noted—as expected—that degradation of DNA into ever shorter fragments begins rapidly after death by action of the body’s own enzymes, and then by action of enzymes from microorganisms. The overall rate of decay of DNA is also influenced by environmental conditions are such as pH and, of course, temperature, as shown in the following graph, which was entitled ‘survival of the coldest.’

 

plot

Plot of the rate of DNA decay vs. temperature for estimated half-lives of 30- and 100-base-pair (bp) DNA fragments. The estimated ages and temperatures of material used to recover the genomes of a Neanderthal (N), a woolly mammoth (M) and the horse fossil discovered at Thistle Creek, Canada (H) are shown [C. D. Millar & D. M. Lambert, Nature, Vol. 499, pp. 34-35 (2013)].

In closing this blog, I encourage you to get an appreciation for the impressive technical depth and scientific breadth of this conference by taking a look at the list of presentation titles and abstract, if you haven’t done that already.  NGS has truly revolutionized multiple and diverse fields of basic science and enabled a seemingly never-ending series of new and improved applications. Among these are studies of metagenomics and microbiomes, which will be the subject of this blog in the near future.

As always, your comments are welcomed.