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 8^th, 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.”

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.

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 3^rd 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.

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™.

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 8^th 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

On October 20^th 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 17^th 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.

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

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 14^th 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.