De-Extinction: Hope or Hype?

  • Can scientists “revive” woolly mammoths?
  • Passenger Pigeons, possibly?
  • Is “facilitated adaption” more realistic?

If you haven’t seen the 1993 movie Jurassic Park, the plot involves a tropical island theme park populated with cloned dinosaurs created by a bioengineering company, InGen. The cloning was accomplished by extracting the DNA of dinosaurs from mosquitoes that had been preserved in amber—not unlike extraction of ancient yeast DNA from extinct bees preserved in amber for brewing “Jurassic beer” that I featured in a previous posting. However, in Jurassic Park the strands of DNA were incomplete, so DNA from frogs was used to fill in the gaps. The dinosaurs were cloned genetically as females in order to prevent breeding.

This is all a great premise for a movie, but will Jurassic Park-like fantasy become reality in the near future?  What’s being investigated now, and are there concerns being voiced? These are just some of the questions touch upon below.

Woolly Mammoths May One Day Roam Real-Life Jurassic Park

Hendrik Poinar, Director of the Ancient DNA Centre at McMaster University in Hamilton, Ontario (taken from fhs.mcmaster.ca via Bing Images).

Dr. Hendrik Poinar, Director of the Ancient DNA Centre at McMaster University (taken from fhs.mcmaster.ca).

Dr. Hendrik Poinar, Associate Professor at McMaster University in Canada, was trained as a molecular evolutionary geneticist and biological anthropologist, and now specializes in novel techniques to extract and analyze “molecular information (DNA and/or protein sequences)” from ancient samples. His work included such projects as sequencing the mitochondrial genome of woolly mammoths that went extinct long ago. Based on that work, Dr. Poinar was recently interviewed by CBC News about the likelihood of reestablishing woolly mammoths. Here are some excerpts:

Q: Without getting too technical, describe what you’re doing to bring back animals like the woolly mammoth?

A: We’re interested in the evolutionary history of these beasts. These lumbering animals lived about 10,000 years ago and went extinct. We’ve been recreating their genome in order to understand their origins and migrations and their extinction. That led to the inevitable discussion about if we could revive an extinct species and is it a good thing.

Q: Why is this so interesting to you?

A: There are reasons why these animals went extinct. It could be climate, it could be human-induced over-hunting. If we can understand the processes that caused extinction, maybe we can avoid them for current endangered species. Maybe we need to think about what we can do to bring back extinct species and restore ecosystems that are now dwindling.

Q: Is it possible to bring these things back to life?

A: Not now. We’re looking at 30 to 50 years.

Woolly mammoths roamed both North America and Asia for hundreds of thousands of years. Many went extinct during the most recent period of global warming (taken from CBC News via Bing Images).

Woolly mammoths roamed both North America and Asia for hundreds of thousands of years. Many went extinct during the most recent period of global warming (taken from CBC News via Bing Images).

Q: How would you do something like that?

A: First thing you have to do is to get the entire blueprint. We have mapped the genome of the woolly mammoth. We’re almost completely done with that as well as a couple other extinct animals. We can look at the discrete differences between a mammoth and an Asian elephant. We would take an Asian elephant chromosome and modify it with mammoth information. Technology at Harvard can actually do that. Take the modified chromosomes and put them into an Asian elephant egg. Inseminate that egg and put that into an Asian elephant and take it to term. It could be as soon as 20 years.

Q: Is this such a good idea?

A: That’s the million-dollar question. We’re not talking about dinosaurs. We’ll start with the herbivores—the non-meat eaters. We could use the technology to re-introduce diversity to populations that are dwindling like the cheetah or a wolf species we know are on the verge of extinction. Could we make them less susceptible to disease? Is it good for the environment? We know that the mammoths were disproportionately important to ecosystems. All the plant species survived on the backs of these animals. If we brought the mammoth back to Siberia, maybe that would be good for the ecosystems that are changing because of climate change.

Q: You are tinkering with the evolutionary process?

A: Yes, but would you feel differently if the extinction was caused by man like it was with the passenger pigeon or the Tasmanian wolf, which were killed by humans? Even the large mammoth, there are two theories on their extinction, one is overhunting by humans…and the other is climate. Do we have a moral obligation?

Bringing Back Passenger Pigeons

Ben Novak has a BS in Ecology and worked with mastodon fossils toward a master’s degree at McMaster University, but he abandoned that to pursue his long-time passion for passenger-pigeon genetics (taken from wfs.org via Bing Images).

Ben Novak has a BS in Ecology and worked with mastodon fossils toward a master’s degree at McMaster University, but he abandoned that to pursue his long-time passion for passenger-pigeon genetics (taken from wfs.org via Bing Images).

Ben Novak, according to an interview in Nature last year, has spent his young career endeavoring to resurrect extinct species. Although he has no graduate degree, he has amassed the skills and funding to start a project to bring back the Passenger Pigeon—once the United States’ most numerous bird (about 5 billion according to Audubon)—which died out in 1914. Following are comments from Ben, taken from the Nature article referenced above, about how his work is funded and its prospects.

“Once I had passenger-pigeon tissue [from the Field Museum of natural History in Chicago, Illinois], I started applying for grants to do population analysis, but I couldn’t secure funding. I got about $4,000 from family and friends to sequence the DNA of the samples. When I got data, I contacted George Church, a molecular geneticist at Harvard Medical School in Boston, Massachusetts, who was working in this area. He and members of Long Now Foundation in San Francisco, California, which fosters long-term thinking, were planning a meeting on reviving the passenger pigeon….The more we talked, the more they discovered how passionate I was. Eventually, Long Now offered me full-time work so that nothing was standing in my way.”

“I have just moved to the University of California, Santa Cruz, to work with Beth Shapiro. She has her own sample of passenger pigeons, and we want to do population genetics and the genome. It’s a good fit. Long Now pays me, and we do the work in her lab, taking advantage of her team’s expertise in genome assemblies and ancient DNA.”

Male passenger pigeon (taken from swiftbirder.wordpress.com via Bing Images).

Male passenger pigeon (taken from swiftbirder.wordpress.com via Bing Images).

For the sad story of how this creature went extinct, click here to access an account written by Edward Howe Forbush in 1917.

Doing more searching about Ben Novak led me to another 2013 interview, this time in Audubon. When asked if it’s realistic to get a healthy population from a few museum specimens, here’s what he said.

“If we’re willing to create one individual [passenger pigeon], then through the same process we can produce individuals belonging to completely different genetic families. We can make 10 individuals that, when they’re mated, will have an inbreeding coefficient near zero…First we need to discern what the actual genetic structure of the species was. We can analyze enough tissue samples to get that genetic diversity.”

While perusing the Long Now Foundation’s website, I was pleased to read a Passenger-Pigeon progress report posted by Ben Novak on October 18th 2013.  The posting gives a detailed update on genomic sequencing of “Passenger Pigeon 1871″ [date of preservation] at the University of California San Francisco‘s Mission Bay campus sequencing facility, as well as some nice pictures. Given what he said above about 10 individuals being theoretically adequate for reviving and restoring an extinct population, you’ll be as pleased as Ben is about the following.

“Passenger Pigeon 1871 was selected as the candidate for the full genome sequence for its superb quality compared to other passenger pigeon specimens. Over the last two years Dr. Shapiro, myself and colleagues have scrutinized the quality of 77 specimens including bones and tissues. Our first glimpses of data confirmed that the samples would be able to provide the DNA needed for a full genome sequence, but as we delved into the work, the specimens exceeded our expectations. Not only do we have one specimen of high enough quality for a full genome, we have more than 20 specimens to perform population biology research with bits of DNA from all over the genome.”

Revive and Restore

Reading about Ben Novak’s support from the Long Now Foundation led me discover the organization’s Revive and Restore Project, aimed at genetic rescue of endangered and extinct species. Its mission is stated as follows:

“Thanks to the rapid advance of genomic technology, new tools are emerging for conservation. Endangered species that have lost their crucial genetic diversity may be restored to reproductive health. Those threatened by invasive diseases may be able to acquire genetic disease-resistance.

It may even be possible to bring some extinct species back to life. The DNA of many extinct creatures is well preserved in museum specimens and some fossils. Their full genomes can now be read and analyzed. That data may be transferable as working genes into their closest living relatives, effectively bringing the extinct species back to life. The ultimate aim is to restore them to their former home in the wild.

Molecular biologists and conservation biologists all over the world are working on these techniques. The role of Revive and Restore is to help coordinate their efforts so that genomic conservation can move ahead with the best current science, plenty of public transparency, and the overall goal of enhancing biodiversity and ecological health worldwide.”

This Project’s website is well worth visiting, as it provides a fascinating mix of species under consideration (such as the Passenger Pigeon and the woolly mammoth), various video presentations by advocates, and an engaging blog. It also provides a very convenient “donate” button should you be so inclined.

While the Passenger Pigeon project and other Revive and Restore efforts are well intended, I’m more inclined at this time to be neutral-to-negative about the projects, and will reserve a final opinion until all parties, pro and con, have extensive debates similar to what was done in the past for then (and still) controversial recombinant DNA technology. Given the amount of concern and caution then for what we can now view as conventional genetic engineering, it seems reasonable to me that, with far more powerful tools for genomics and synthetic biology being available, “an abundance of caution” is in order when dealing with the possibility of resurrecting extinct species. If Jurassic Park serves as any sort of model for what science can accomplish, perhaps we should also consider what the movie highlights as the potential implications of those accomplishments.

For now, I’m intently interested in the continuing debates and I find it fascinating to consider alternatives such as rescuing species from extinction as outlined next.

“Facilitated Adaption” Pros & Cons

Michael A. Thomas, Professor of Biology at Idaho State University, and colleagues authored a Comment in Nature last year entitled Gene tweaking for conservation that is freely available (yeh!) and well worth reading. Some highlights are as follows:

Sadly, if not shockingly, conservative estimates predict that 15–40% of living species will be effectively extinct by 2050 as a result of climate change, habitat loss and other consequences of human activities. Among the interventions being debated, facilitated adaptation has been little discussed. It would involve rescuing a target population or species by endowing it with adaptive alleles, or gene variants, using genetic engineering—not too unlike genetically modified crops that now occupy 12% of today’s arable land worldwide. Three options for facilitated adaption are outlined.

“Poster Child” for facilitated adaption: an endangered Florida panther population was bolstered through hybridization with a related subspecies — a technique that could be refined using genomic tools (taken from Thomas et al. Nature 2013).

“Poster Child” for facilitated adaption: an endangered Florida panther population was bolstered through hybridization with a related subspecies — a technique that could be refined using genomic tools (taken from Thomas et al. Nature 2013).

First, threatened populations could be cross with individuals of the same species from better-adapted populations to introduce beneficial alleles. A good example of this is crossing a remnant Florida panther population with related subspecies from Texas that significantly boosted the former population and its heterozygosity, a measure of genetic variation that was desired. Risks of this approach include dilution of locally adaptive alleles.

Second, specific alleles taken from a well-adapted population could be spliced into the genomes of threatened populations of the same species. This was exemplified by recent work wherein heat-tolerance alleles in a commercial trout were identified for possible insertion into fish eggs in populations threatened by rising water temperature. Such an approach was viewed as low risk because it involves genetic manipulations within the same species.

Third, genes removed from a well-adapted species could be incorporated into the genomes of endangered individuals of a different species. This transgenic approach has been extensively used to improve plant crops toward drought and temperature. However, outcomes are hard to predict, and a major concern is that such an approach could bring unintended and unmanageable consequences—definitely a scary possibility.

What do you think about reintroducing extinct species?  Do you see other pros and cons to facilitated adaption?  As always, your comments are welcomed.

Postscript

The following, entitled ‘De-Evolving’ Dinosaurs from Birds, recently appeared in GenomeWeb:

Ancient animals could be resurrected through the genomes of their modern-day descendants, Alison Woollard, an Oxford biochemist tells the UK’s Daily Telegraph. For instance, the DNA of birds could be “de-evolved” to resemble the DNA of dinosaurs, the paper adds.

“We know that birds are the direct descendants of dinosaurs, as proven by an unbroken line of fossils which tracks the evolution of the lineage from creatures such as the velociraptor or T-Rex through to the birds flying around today,” Woollard says, later adding that “[i]n theory we could use our knowledge of the genetic relationship of birds to dinosaurs to ‘design’ the genome of a dinosaur.”

In both the book and movie Jurassic Park, the fictional resurrection of dinosaurs relied on dinosaur DNA that was preserved in fossilized biting insects, but as the Daily Telegraph notes, a study in PLOS One earlier this year found no evidence of DNA from amber-preserved insects.

Daily Telegraph adds that any dinosaur DNA recovered from bird genomes would be fragmented and difficult to piece back together. A mammoth, it says, might have a better shot.

Mixed Coupling: “Bite-sized” Topics of Interest

  • What do DREAMing, King Louis XVI, Sasquatch, Abominable Snowman, and “Jurassic Beer” have in common?
  • Hint:  Sequencing

Keeping up with current literature in order to select items to weave into my blog content is greatly assisted by taking advantage of automated alerts. My daily alerts from PubMed and Google Scholar are based on keywords, such as nucleoside/tide analogs, oligonucleotides, sequencing, modified mRNA, etc. Not surprisingly, I come across many interesting or useful articles that are “content-worthy” but not as an entire or lengthy posting. So, today’s content represents a collection of these “bite-sized” topics that I refer to as “mixed couplings”—a termed borrowed from my early publication on analysis of mixed-sequence oligonucleotides derived from mixed coupling of phosphoramidite reagents.

While today’s mixture of topics are loosely “coupled” by virtue of involvement of sequencing, in one form or another, future mixed-coupling content will incorporate other technical aspects of what’s trending in nucleic acid research, development or applications. Hopefully you will find these to be as interesting or informative as I do.

DREAMing…of a patent-free human genome for clinical sequencing

This is the catchy and provocative title of an article in the October 8th, 2013 online issue of Nature Biotechnology by Kevin McKernan et al. at Courtagen Life Sciences. The article  proposes a novel approach for using the genetic code without concern for existing gene patents. They state that, “[i]n our opinion, gene patents are immoral not because of a profit motive but because an ‘armed authority’ ultimately enforces them to protect a piece of ‘imagined property’ or an idea. Property is usually defined as something that consists of matter and is not infinitely replicable (i.e. exhibits scarcity). Ideas are neither of these.”

Kevin McKernan, co-founder, CEO and Director of Courtagen Life Sciences, Inc., which provides innovative genomic and proteomic products and services for physicians and the Life Sciences industry (taken from courtagen.com via Bing Images).

Kevin McKernan, co-founder, CEO and Director of Courtagen Life Sciences, Inc., which provides innovative genomic and proteomic products and services for physicians and the Life Sciences industry (taken from courtagen.com via Bing Images).

The entire legal rationale and technical underpinning of the proposed methodology in this article are not easily summarized, should be read carefully, and thought about deeply. Nevertheless, some of the key points are as follows.

Despite the Supreme Court’s recent decision on the patent ineligibility of natural DNA, cDNAs—a human-made laboratory modification—remain patentable, provided other legal considerations are met. If cDNAs are human-made, PCR amplification of gene panels and exomes would also constitute human-manipulated isolation of the claimed DNA sequence. However, this isolation via amplification significantly alters the DNA by failing to faithfully replicate the molecular epigenetic (i.e. 5-methyl CpG and other minor dinucleotide) patterns in natural DNA. Most gene patents fail to specify methylated sequence IDs. As a result, the impact of methylation on sequence function and utility is of paramount importance in this matter.

The argument continues by reasoning that any composition-of-matter claim to a sequence ID would have to pass a triple identity test: does methylated sequence perform substantially the same function, in the same way to yield the same result as non-methylated sequence. They then provide various reasons for why this argument of equivalence is becoming harder if not impossible to make, and illustrate this point by carrying out Agilent Haloplex v2 target enrichment PCR to sequence 327 genes with varying concentrations (0.05mM – 0.2mM) of 5-methyl-dCTP added to the PCR cocktail. This technique replicates natural CpG and other minor dinucleotide patterns, but also introduces non-CpG methylation, thus producing amplicons even more distant from the claimed 4-nucleotide sequences than the natively methylated versions of the gene not described in most gene patents.

As expected, subtle amplicon mobility shifts and non-identical Illumina MiSeq results were obtained, yet with 50mM 5-methyl-dCTP 99.5% of the sequence information matched the control data set obtained without 5-methyl-dCTP.

A methylation-specific restriction enzyme, MspJI, is employed in this method to digest background 5-methyl-dCTP generated amplicons, not unlike use of UNG and dUTP for “carryover decontamination”. Consequently, they coined the term DREAM PCR for Decontamination Ready Encoded AMplification to describe a PCR method that utilizes additional 5-methyl-dCTP to generate an amplicon set that is susceptible to methylation-specific digestion. This aspect of DREAMing is said to provide additional assurances to clinical sequencing laboratories.

My last check on the metrics for this Nature Biotechnology article indicated that it’s in the 95th percentile of a sample of 10,000 of the 57,603 tracked articles of a similar age in all journals. There were also more than 50 tweets so I expect that it will continue to generate lots of commentary in social media, as well as much controversy, and countless billable hours by patent and litigation attorneys!

Louis XVI’s blood in a gourd?

Application of modern methods for DNA analysis to identify historical individuals’ remains is a fascinating subject that has included analysis of the remains of the Romanov family, the putative evangelist Luke, the American outlaw Jesse James, and the astronomer Nicolaus Copernicus. Now add the French King Louis XVI—and circumstances somewhat reminiscent of a Dan Brown novel, legends about the Holy Grail, or the Shroud of Turin.

Louis XVI became the heir to the throne and the last Bourbon king of France upon his father’s death in 1765. In 1770, he married Austrian archduchess Marie-Antoinette, the daughter of Maria Theresa and Holy Roman Emperor Francis I. After a slew of governing missteps, Louis XVI brought the French Revolution crashing down upon himself, and on January 21, 1793 he was executed.

Displaying the head of Louis XVI-1793 (taken from robinengelman.com via Bing Images).

Displaying the head of Louis XVI-1793 (taken from robinengelman.com via Bing Images).

According to a publication in Forensic Science entitled Genetic analysis of the presumptive blood from Louis XVI, king of France by Lalueza-Fox et al., eyewitnesses stated that many people from the crowd dipped their handkerchiefs in the king’s blood and kept these objects as mementos. For more than one hundred years, an Italian family has owned an ornate desiccated gourd—of a type used to store gunpowder—that presumably contained one of these handkerchiefs. An inscription read “Maximilien Bourdaloue on January 21th, dipped his handkerchief in the blood of the king after his beheading”. Biochemical analyses of a dark, dried spot in the gourd confirmed that the sample was indeed blood. On April 3rd this year the bloodstained cloth was sold at auction for $24,000 to a French collector who is fascinated by the former monarch, according to an online news report.

Gourd containing handkerchief thought to be stained with blood of Louis XVI (taken from u.tearn.com via Bing Images).

Gourd containing handkerchief thought to be stained with blood of Louis XVI (taken from u.tearn.com via Bing Images).

A report entitled Genetic analysis of the presumptive blood from Louis XVI, king of France was subsequently published by a consortium of Spanish and Italian investigators in Forensic Science International: Genetics. This report by Lalueza-Fox et al., which can be freely downloaded via Google Scholar, provides details for how several samples were scraped from the inside of the gourd for extraction of presumptive ancient DNA with standard precautions to avoid contamination. Various DNA analyses were carried out, including PCR-sequencing of mitochondrial DNA (mtDNA) hypervariable region 1 (HV1) and 2 (HV2)—see TriLink’s website for mtDNA analysis and its recently launched mitoPrimers™.

Crystal urn believed to contain the heart of Louis XVII (taken from flickriver.com via Bing Images)

Crystal urn believed to contain the heart of Louis XVII (taken from flickriver.com via Bing Images)

In addition, because Louis XVI had blue eyes, as can be seen in different portraits, these investigators checked a single-nucleotide polymorphism (SNP; rs12913832) that is associated with blue eye color in modern humans, and is located in exon 86 of the HERC2 gene. These results showed that the subject analyzed was a heterozygote, which is compatible with a blue-eyed person. The investigators concluded that to confirm the identity of the subject, an analysis of the dried heart of his son, Louis XVII, could be undertaken. That too is an interesting “story within a story” worth reading by clicking here, especially if you’re into mixing modern forensics with old rumors, mystery and controversy involving royals. According to official historiography, the 10-year-old Louis XVII died in the Temple of Paris on June 8th 1795. However, public rumor spread the theory that Louis XVII escaped and that his descendants would be alive today. One such putative “Louis XVII” was Carl Wilhelm Naundorff, who died in 1845 in Delft, Netherlands. Comparative mtDNA analysis was performed on the heart of the young boy who died in the prison of Paris in 1795.

Sasquatch sequencing

Sasquatch or hoax? (taken from spatzo.net via Bing Images)

Sasquatch or hoax? (taken from spatzo.net via Bing Images)

On October 20th 1967, Roger Patterson and Robert Gimlin recorded this now famous picture of a purported Sasquatch with a 16mm camera at Bluff Creek, California, after large footprints had been found in this region in previous years. Many years later, Bob Heironimus, an acquaintance of Patterson’s, claimed that he had worn an ape costume for the making of the film. Both men have always dismissed allegations that they had hoaxed the footage by filming a man wearing an ape suit.

Fast forward from 1967 to this recent 2013 press by the Sasquatch Genome Project, website I highly recommend visiting for much more information and access to links to various videos:

DALLAS, Oct 1 – On October 1, the Sasquatch Genome Project held a news conference in Dallas to show exclusive footage from the long-awaited Erickson Project, a multi-site effort led by entrepreneur Adrian Erickson to capture definitive video and DNA evidence from the elusive Sasquatch. Along with Erickson participants in the genome project spoke about their areas of expertise and answered reporters questions.

The Sasquatch Genome Project, led by Dr. Melba Ketchum, is the group responsible for the 5-year study and genomic sequencing of Sasquatch DNA, “Novel North American Hominins, Next Generation Sequencing of Three Whole Genomes and Associated Studies,” that passed scientific peer review in January and was published in February of this year. In conjunction with the screening of the new Erickson footage, the DNA study is available for on-line open access on this web site under the tab View DNA Study.

Adrian Erickson presented short clips from his HD footage. Researcher Dennis Pfohl, who personally captured video and collected DNA samples from Sasquatch individuals spoke about the footage and the project. Dr. Ketchum presented physical Sasquatch samples used in the DNA study and new specimens under ongoing investigation, and she also discussed sample chain of custody, study results, and bias encountered from the scientific establishment.

Lest you immediately dismiss the Sasquatch Genome Project and this publication as a modern version of a continuing hoax or scam, you should at least give the publication a quick read, and consider the authors’ affiliations, as well as Dr. Ketchum’s CV that are all accessible on the Project website. I’ll briefly mention here some aspects of the publication.

One hundred and eleven samples of blood, tissue, hair, and other types of specimens were studied. DNA from a subset of these samples gathered from various locations in North America that survived screening for wildlife species identification were subjected to mtDNA sequencing, specific genetic loci sequencing, forensic STR testing, whole genome SNP analysis, and NGS genome sequencing. The authors conclude the “the data conclusively proves that the Sasquatch exists as an extant hominin and are a direct maternal descendent of modern humans. At this time, analysis of the Sasquatch genomes is still ongoing…Additionally, analysis of hair purportedly from a Siberian Wildman is being tested in an effort to determine if relatedness between the Sasquatch and the Russian Wildmen. A species name has been applied for with ZooBank, Homo sapiens cognatus.” Online English translations for the Latin word cognatus are related by blood, a relative, kinsman.

By the way, the Sasquatch Genome Project homepage provides a perhaps not surprising account of difficulties encountered in trying to publish this investigation. After various rejected submissions, a journal agreed to publish the reviewed manuscript, but its legal counsel advised against that for such a controversial subject as it would destroy the editors’ reputations. Rather than spend another five years just trying to find a journal to publish, rights to this journal were acquired and it was renamed to Denovo, but retained the passing peer reviews that are expected by the public and the scientific community.

Abominable Snowman (aka Yeti) Too?

According to an Oct 17th headline for The Telegraph in the U.K., ‘Yeti lives’: Abominable Snowman is ‘part polar bear and still roams the Himalayas’. The article states that research by Bryan Sykes, who is founder and chairman of Oxford Genetics, a genealogical DNA testing firm, and a professor of human genetics at the University of Oxford, has found a genetic match between an ancient polar bear and samples said to come from the Yeti—suggesting the creature known as the Abominable Snowman is still living in the Himalayas.

Sykes conducted DNA tests on hairs from two unidentified animals, one found in the western Himalayan region of Ladakh, in northern India, and the other from Bhutan, 800 miles east. The results were then compared with other animals’ genomes stored on a database of all published DNA sequences. Sykes found a 100 percent match with a sample from an ancient polar bear jawbone found in Svalbard, Norway. That specimen dates back at least 40,000 years, and probably as far back as 120,000 years—a time when the polar bear and the closely related brown bear were separating as different species.

Sykes believes that the animals are hybrids—crosses between polar bears and brown bears. Because the newly identified samples are from creatures which are recently alive, he thinks the hybrids are still living in the Himalayas. He added: “There’s more work to be done on interpreting the results. I don’t think it means there are ancient polar bears wandering around the Himalayas. But we can speculate on what the possible explanation might be. It could mean there is a sub species of brown bear in the High Himalayas descended from the bear that was the ancestor of the polar bear. Or it could mean there has been more recent hybridisation between the brown bear and the descendant of the ancient polar bear.”

Jurassic beer

Given the prestige and widespread readership of Science magazine, it’s not surprising that a lot of attention was given to an article therein by Cano & Borucki in 1995 reporting to have extracted, revived, cultured and identified bacterial spores from the abdominal contents of extinct Proplebeia dominicana bees. The bees were preserved in 25- to 40-million-year-old amber—a polymeric glass formed over time from resins of conifers and plants that provides an excellent preservative matrix. Interest in this amazing report was undoubtedly due to its appearance shortly after the famously popular movie Jurassic Park, with a plot that revolved around cloning a dinosaur from fossilized DNA in dinosaur blood from mosquitos trapped in amber.

Extinct Proplebeia dominicana bee in amber (taken from pinterest.com via Bing Images)

Extinct Proplebeia dominicana bee in amber (taken from pinterest.com via Bing Images)

In a later patent application by Dr. Cano, he describes a novel yeast strain recovered and cultured from a 44-million-year-old piece of amber that he said is “similar to Saccharomyces…and may be used in the manufacture of a fermented beverage (e.g. beer). Surprisingly, in the manufacture of beer, the yeast strain exhibits properties that make it amenable to the manufacture of both a lager and ale.” Dr. Cano now co-owns Fossil Fuel Brewing Co. which is utilizing ancient yeast strains to brew beer. Jay R. Brooks, the tasting director of the

Jurassic Beer (taken from mutineermagazine.com via Bing Images

Jurassic Beer (taken from mutineermagazine.com via Bing Images

exalted Celebrator Beer News Magazine, commented when comparing the Fossil Fuels brew to an identical pale ale differing only in the strain of yeast, “[Fossil Fuels] is smoother, with softer fruity flavor characteristics and just a touch of lemony sweetness that isn’t tart…It has a more complex and well-developed taste profile, and its smoothness makes it great. The fact it is made with such old yeast is fascinating, and given how good the beer is, no mere novelty.”

Caveat on reproducibility

Two years after the aforementioned Science publication by Cano & Borucki in 1995, Austin et al. at the National History Museum in London, U.K. reported rigorous attempts to reproduce these DNA sequences using the same amber sample, as well as others. The only sequences they were able to detect were derived from obvious sources of non-insect contamination. They concluded that “although no negative result can disprove the existence of ancient DNA in amber-preserved fossils, our work shows that isolation of geologically ancient DNA from amber-preserved insects is not reproducible”.

This non-reproducibility was also reported in PLoS ONE in September 2013 by a team of scientists at the University of Manchester in the U.K.  Using high-throughput (NGS) sequencing methods, they concluded that they “were unable to obtain any convincing evidence for the preservation of endogenous DNA in either of the two copal [aka “young amber”] inclusions that [they] studied”. The investigators reasoned that their negative results could not be attributed solely to a lack of technical skill, as they have successfully isolated and sequenced ancient Mycobacterium tubercolosis DNA from human bones as well as DNA from archaeological plant samples, nor to their extraction and preparation methods as those approaches have been used to isolate DNA from air-dried specimens. A GenomeWeb story about this publication quoted one coauthor as saying that “[w]e therefore conclude that our failure to obtain sequence reads was because the copal specimens contained no preserved DNA,” while another coauthor noted that “unfortunately, the Jurassic Park scenario must remain in the realms of fiction”.

But hold on, it’s important to consider the following comment about the aforementioned PLoS ONE publication that is online and reads as follows:

Should be titled “Absence of DNA sequenceable by 454”

Posted by John Thompson on 14 Sep 2013 at 14:31 GMT 

I have no doubt that this work was carefully done and I have no reason to doubt the results. The conclusions, however, are over-reaching. Having no DNA that is sequenceable by 454 (which requires amplification and relatively long, intact, unmodified DNA) is not at all the same as having no DNA. There are examples of DNA that cannot be sequenced by 454 but can be detected by single-molecule methods. A proper study of DNA survivability in amber will require the most sensitive assays and not just the most accessible. Is it likely that Jurassic era DNA has survived in amber? No, but this work does not prove it.

I contacted John Thompson, former Senior Director of Genomic Research at Helicos BioSciences, who kindly agreed to identify himself as having posted the above comment, with which I fully agree. He also added the following:

The two samples I worked with that were completely or initially immune to Illumina and 454 sequencing were the ancient horse DNA recently published in Nature (499: 74-78, doi:10.1038/nature12323, “Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse.” and DNA extracted from remains of Korean war MIAs (http://www.ashg.org/2009meeting/abstracts/fulltext/f21866.htm). The former sample was eventually sequenced by Illumina once Helicos had identified how to best isolate the DNA (see references in Nature paper). The Korean War MIA data was never published beyond the abstract because the informed consent was not broad enough to cover what Helicos sequenced. AFDIL was attempting to get 40 bp of mitochondrial sequence but we actually got the entire mitochondrial genome and could have gotten most of the nuclear genome if we had kept going. Illumina and 454 could not always get the 40 bp.

Given this unique performance of Helicos single-molecule sequencing, it’s worth mentioning that fee-for-service Helicos sequencing is available, as detailed at http://www.seqll.com/.

Ah, how interesting our ever-changing field is! I hope you enjoyed this mixed coupling of bite-size topics and I encourage you to share your own with all readers via a post below. 

Postscript

Shortly after finishing this posting, I read the following October 14th online Nature headline, byline and story written by Ed Yong that I felt compelled to add as this postscript:

Blood-filled mosquito is a fossil first

Insect’s bloated abdomen carries traces of blood molecules that are 46-million-years old.

Jurassic Park’s iconic image of a fossilized blood-filled mosquito was thought to be fiction—until now. For the first time, researchers have identified a fossil of a female mosquito with traces of blood in its engorged abdomen. A team led by Dale Greenwalt at the US National Museum of Natural History in Washington DC reports the fossil discovery today in Proceedings of the National Academy of Sciences.

Although scientists have found fossils of suspected blood-sucking insects, the creatures’ feeding habits have mostly been inferred from their anatomy or the presence of blood-borne parasites in their guts. But Greenwalt’s fossilized mosquito contains molecules that provide strong evidence of blood-feeding among ancient insects back to 46 million years ago. It is a fortunate find. “The abdomen of a blood-engorged mosquito is like a balloon ready to burst. It is very fragile,” says Greenwalt. “The chances that it wouldn’t have disintegrated prior to fossilization were infinitesimally small.”

A long shot

The insect was found not in amber, as depicted in Jurassic Park, but in shale sediments from Montana. After 46 million years, any DNA would be long degraded, but other molecules can survive. Greenwalt’s team showed that the insect’s abdomen still contains large traces of iron and the organic molecule porphyrin — both constituents of haemoglobin, the oxygen-carrying pigment found in vertebrate blood. These molecules were either rare or absent in the abdomen of a fossilized male mosquito (which does not drink blood) of the same age, found at the same location.

“This shows that details of a blood-sucking mosquito can be nicely preserved in a medium other than amber,” says George Poinar, who studies fossilized insects at Oregon State University in Corvallis. “It also shows that some porphyrin compounds in vertebrate blood can survive under the right conditions for millions of years.”

Greenwalt suggests that this provides support for the controversial claims of Mary Schweitzer, a palaeontologist at North Carolina State University in Raleigh, who has reportedly isolated haemoglobin traces from dinosaur bones.

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.