Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics

  • Invasive Needles and Scalpels Seen as Passé
  • Noninvasive Sampling Advocates Focusing on Circulating Tumor Cells (CTCs) 
  • New Companies are Pursuing the Liquid Biopsy “Gold Rush”

Biopsy Basics

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

As defined in Wikipedia, a biopsy is ‘a medical test commonly performed by a surgeon or an interventional radiologist involving sampling of cells or tissues for examination.’ Biopsies can be excisional (removal of a lump or area), incisional (removal of only a sample of tissue), or a needle aspiration (tissue or fluid removal). Despite the value of these traditional types of biopsies, they are more or less invasive, lack applicability in certain instances, and require accurately “going to the source” of concern, as pictured to the right, for ultrasound-guided breast cancer biopsy. Better methodology is highly desirable and is the topic of this post. By the way, if you want to peruse a lengthy list of scary risks associated with various type of common invasive biopsies, click here to see what I found in Google Scholar by searching “incidence of complications from biopsies.”

Continue reading

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

Children at Risk from Deadly Respiratory Virus EV-D68

  • Frightening Statistics From CDC
  • CDC Updated U.S. Map of Outbreak & Advice of What to be Aware
  • CDC Develops Rapid Real-Time RT-PCR Test for Detection
  • Some Speculate on Linking Outbreak to the “Southern Border Invasion”

Ebola virus is dominating news reports lately, and perhaps rightly so considering the worldwide impact. Turning our attention, however, to actual incidents of infection and death in the U.S., enterovirus (EV) D68 poses a much greater threat and warrants our attention—especially if you or your friends have young children.

On September 24, Eli Waller’s parents were worried that their 4 year-old son had pink eye and kept him home from school so that he wouldn’t infect other children. He seemed otherwise healthy. What happened next was shocking.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

‘He was asymptomatic and fine, and the next morning he had passed,’ said Jeffrey Plunkett, the township’s health officer. ‘The onset was very rapid and very sudden,’ quoted the NY Times.

A week later the Centers for Disease Control and Prevention (CDC) confirmed that Eli had been infected with EV-D68.

EV-D68 was seen as early as August of this year as hospitals in Missouri and Illinois reported increased visits from children with respiratory illness. Soon, the virus was identified in 43 states and detected in 594 patients, 5 of which died.

After reading this very sad—if not frightening—story, I decided to research EV-D68 for this “hot topic” blog, which I’m dedicating to little Eli Waller.

Continue reading

Sequence Every Newborn?

  • Envisaged in the 2002 Challenge for Achieving a $1,000 Genome
  • Are We There Yet? Yes…and No
  • So Where Are We, Actually?

The notion of metaphorically ‘dirt cheap’ genome sequencing is now so prevalent that it seems to have been always available—and have virtually unlimited utility—as previously touted in a provocative article in Nature Biotechnology rhetorically entitled What would you do if you could sequence everything? The notion of ‘everything’ obviously includes all people, and—as we’re reminded by the now familiar t-shirt statement—‘babies are people too.’ Unlike the ‘big bang’ origin of the universe, cheap-enough-sequencing-for-everything, including all babies (newborns, actually) just didn’t happen spontaneously, so when and how did this come about?

In the Beginning…

According to the bible, the history of creation began when, “in the beginning God created the heavens and the earth.” The history of cheap-enough-sequencing-for-everything, according to my tongue-in-cheek reckoning, is that “In the beginning Craig Venter convened and moderated a diverse panel of experts to discuss The Future of Sequencing: Advancing Towards the $1,000 Genome.” This was a ‘hot topic’ at the 14th International Genome Sequencing and Analysis Conference (GSAC 14) in Boston on Oct 2nd 2002. The pre-conference press release added that “the panel will explore new DNA sequencing technologies that have the potential to change the face of genomics over the next few years.”

While it’s taken considerably more than a ‘few years’ to achieve the $1,000 genome, continual decrease in sequencing costs has enabled large-scale sequencing initiatives such as the 1000 Genomes Project. Nick Loman’s 2013 blog entitled The biggest genome sequencing projects: the uber-list! outlines the 15 largest sequencing projects to date and takes a look at some of the massive projects that are currently in the works.

According to GenomeWeb on Jan 14th 2014, Illumina launched a new sequencing system that can now produce a human genome for under $1,000, claiming to be the first to achieve this ‘long sought-after goal’—roughly 12 years in the making, by my reckoning. A LinkedIn post on Jan 15th by Brian Maurer, Inside Sales at Illumina, quotes Illumina’s CEO as saying that “one reagent kit to enable 16 genomes per run will cost $12,700, or $800 per genome for reagents. Hardware will add an additional $137 per genome, while sample prep will range between $55 and $65 per genome.” While Illumina is staking claim to the $1,000 genome and customers acknowledge the new system provides a drastic price reduction, the actual costs of sequencing a genome are being debated. Sequencing service provider, AllSeq, discusses the cost breakdown in their blog. While initially a bit negative, their outlook on the attainable costs seems to be improving.

So, with affordable genome sequencing apparently being a reality, are we ready to sequence every newborn?

Yes…and No

Why this conflicting answer of ‘yes…and no’? The ‘yes’ part is based on the fact that NIH has recently funded four studies on the benefits—and risks—of newborn genome sequencing. The ‘no’ part reflects the added facts that these studies will take five years, and will look at how genome testing of newborns could improve screening, and address what some geneticists view as their most sensitive ethical questions yet.

Put another way, and as detailed in the following section, the requisite low-cost DNA sequencing technology is now available but it needs to be demonstrated—through technical feasibility investigations and in clinical pilot studies—that newborns receive health benefits of the type expected, and that numerous ‘tricky’ ethical issues can be dealt with in an ‘acceptable manner’—although to whom it is acceptable is not obvious to me at this time. Likely controversial views on reimbursement also have to be addressed, but that’s a whole other topic—dare I say ‘political football’—vis-à-vis Obamacare, ooops, I mean the Affordable Care Act.

So What’s the Plan?

The following answer is an adaptation of the Sep 13th 2013 Science News & Analysis by Jocelyn Kaiser entitled Researchers to Explore Promise, Risks of Sequencing Newborns’ DNA.

The National Institute of Child Health and Human Development (NICHD) is rolling out a $25 million, 5-year federal research program to explore the potential value of looking at an infant’s genome to examine all of the genes or perhaps a particularly informative subset of them. This genome testing could significantly supplement the decades-old state screening programs that take a drop of blood from nearly every newborn’s heel and test it for biochemical markers for several dozen rare disorders. Diagnosing a child at birth can help prevent irreversible damage, as in phenylketonuria, a mutant-gene metabolic disorder that can be controlled with diet.

Sequence Every Newborn?

Handle with care. Genome testing could enhance newborn screening, but it raises ethical issues [credit: Spencer Grant/Science Vol. 341, p. 1163 (2013)].

Screening for biochemical markers often turns up false positives, however, which genetic tests might help avoid. Moreover, genome sequencing of a single sample could potentially look for all of the ~4,000 (some estimate ~10,000) monogenic diseases—i.e., those caused by defects in single genes. For more information on the subject, the World Health Organization website provides a good introduction.

The ethical concern here is that genome sequencing, unlike the current newborn screening tests, could potentially reveal many more unexpected genetic risks, some for untreatable diseases. Which of these results should be divulged is already controversial, according to Kaiser, who added that “sparks are still flying” over an earlier report described in Science as follows:

Geneticists, ethicists, and physicians reacted with shock to recommendations released last week by the American College of Medical Genetics and Genomics: that patients undergoing genomic sequencing should be informed whether 57 of their genes put them at risk of serious disease in the future, even if they don’t want that information now. The recommendations also apply to children, whose parents would be told even if illness wouldn’t strike until adulthood. The advice runs counter to the long-standing belief that patients and parents have the right to refuse DNA results. This is the first time that a professional society has advised labs and doctors what to do when unanticipated genetic results turn up in the course of sequencing a patient’s genome for an unrelated medical condition.

Given this background, it’s reassuring—in my opinion—that NICHD is taking a ‘go slow’ approach. I’m further reassured that NICHD is funding research of technical and ethical/social issues in four different but interrelated studies (see table below). Kaiser adds that “all will examine whether genomic information can improve the accuracy of newborn screening tests, but they differ in which additional genes they will test and what results they will offer parents.”

New ground: four projects funded at a total of $25 million over 5 years will look at how genome testing could improve newborn screening and other questions [credit: Spencer Grant/Science Vol. 341, p. 1163 (2013)].

New ground: four projects funded at a total of $25 million over 5 years will look at how genome testing could improve newborn screening and other questions [credit: Spencer Grant/Science Vol. 341, p. 1163 (2013)].

More specifically, Stephen Kingsmore at Children’s Mercy Hospital in Kansas City, Missouri, wants to halve the time for his current 50-hour test—discussed in the next section—which he has used to diagnose genetic disorders in up to 50% of infants in his hospital’s neonatal intensive care unit. The test hones in on a subset of genes that may explain the baby’s symptoms. While his group may ask parents if they’re interested in unrelated genetic results, the focus is on ‘a critically ill baby and a distressed family who wants answers,’ Kingsmore told Kaiser.

A team at the University of North Carolina is studying how to return results to low-income families and others who might not be familiar with genomics. It is also dividing genetic findings into three categories—mutations that should always be reported; those that parents can choose to receive, which might include risk genes for adult cancers; and a third set that should not be disclosed, e.g. untreatable adult-onset diseases such as Alzheimer’s.

A team at Brigham and Women’s Hospital in Boston and Boston Children’s Hospital hopes to learn how doctors and parents will use genomic information. ‘We’re trying to imagine a world where you have this information available, whether you’re a sick child or healthy child. How will it change the way doctors care for children?’ asks co-principal investigator Robert Green.

Ethicist Jeffrey Botkin of the University of Utah opined that sequencing might never replace existing newborn screening because of its costs and the complexity, according to Kaiser. However, Kaiser said that Botkin and others believe that it’s important to explore these issues because wealthy, well-informed parents will soon be able to mail a sample of their baby’s DNA to a company to have it sequenced—regardless of whether medical experts think that’s a good idea. ‘There’s an appetite for this. It will be filled either within the medical establishment or outside of it,’ Kaiser quotes Green as saying.

I should add that this parental ‘appetite’ doesn’t seem to be easily satisfied at the moment, based on my—admittedly superficial—survey of what’s currently available in the commercial genome sequencing space. For now, companies such as Personalis and Knome restrict their offerings to researchers and clinicians, not direct-to-consumers, such as parents-on-behalf-of-newborns—yet.

Sample-to-Whole-Genome-Sequencing-Diagnosis in Only 50 Hours

Having been a laboratory investigator during the stunning evolution from manual Maxam-Gilbert sequencing to highly automated Sanger sequencing—the title of this section ‘blows my mind’ and seems impossible—but it’s not! Stephen Kingsmore and collaborators at the Children’s Mercy Hospital in Kansas City, Missouri reported this remarkable achievement in Science Translational Medicine in 2012 and, as noted in the aforementioned table, aim to cut this turnaround time to within 24 hours!

In that 2012 report, they make a compelling case for whole-genome sequence-based diagnostics—and super speedy sample-to-result by noting that monogenic diseases are frequent causes of neonatal morbidity and mortality, and disease presentations are often undifferentiated at birth. Of the ~4,000 monogenic diseases that have been characterized, clinical testing is available for only a handful of them and many feature clinical and genetic heterogeneity. Hence, an immense unmet need exists for improved molecular diagnosis in infants. Because disease progression is extremely rapid—albeit heterogeneous—in  newborns, molecular diagnoses must occur quickly to be relevant for clinical decision-making.

Using the workflow and timeline outlined below, they describe 50-hour differential diagnosis of genetic disorders by whole-genome sequencing (WGS) that features automated bioinformatics analysis and is intended to be a prototype for use in neonatal intensive care units. I should add that an automated bioinformatics analysis is critical for clinical utility, and has been the subject of ‘musings’ by Elaine Mardis in Genome Medicine entitled The $1,000 genome, the $100,000 analysis?

Sequence Every Newborn?Summary of the steps and timing (t, hours) resulting in an interval of 50 hours between consent and delivery of a preliminary, verbal diagnosis [taken from Saunders et al. Sci Transl Med 4, 154ra135 (2012).

To validate the feasibility of automated matching of clinical terms to diseases and genes, they entered retrospectively the presenting features of 533 children, who have received a molecular diagnosis at Children’s Mercy Hospital within the last 10 years, into symptom- and sign-assisted genome analysis (SSAGA)—a new clinico-pathological correlation tool that maps the clinical features of 591 well-established, recessive genetic diseases with pediatric presentations to corresponding phenotypes and genes known to cause the symptoms. Sensitivity was 99.3%, as determined by correct disease and affected gene nominations.

Rapid WGS was made possible by two innovations. First, a widely used WGS platform was modified to generate up to 140 Gb [Gb = giga base pairs = 1,000,000,000 base pairs] of sequence in less than 30 hours (Illumina HiSeq 2500). Secondly, sample preparation took 4.5 hours, while 2 × 100 base pairs of genome sequencing took 25.5 hours. The total ‘hands-on’ time for technical staff was 5 hours.

Readers who are interested in more technical details for sample prep, sequencing, and bioinformatics/analytics should read the full text. However, the authors’ abstract provides the following succinctly described diagnostic ‘payoff’, so to speak:

Prospective WGS disclosed potential molecular diagnosis of a severe GJB2-related skin disease in one neonate; BRAT1-related lethal neonatal rigidity and multifocal seizure syndrome in another infant; identified BCL9L as a novel, recessive visceral heterotaxy gene (HTX6) in a pedigree; and ruled out known candidate genes in one infant. Sequencing of parents or affected siblings expedited the identification of disease genes in prospective cases. Thus, rapid WGS can potentially broaden and foreshorten differential diagnosis, resulting in fewer empirical treatments and faster progression to genetic and prognostic counseling.

These are compelling results, in my opinion. Let me know if you also find this compelling.  As always, your comments are welcomed.

Postscript

After finishing the above post, Andrew Pollack at The New York Times published a fascinating article giving examples of how pharmaceutical companies are heavily investing in genetic studies that employ exome-sequencing of large study groups in a search for clues to aid drug development. Regeneron is conducting one such study that includes 100,000 genomes.

Searching for ‘Genius Genes’ by Sequencing the Super-Smart

  • Brainchild of a high-school dropout
  • Joined by two renowned Professors in the USA and UK
  • Enabled by the world’s most powerful sequencing facility
  • Jonathan Rothberg to do same for math ability 

Prologue

Before plunging into this post, those of you who follow college basketball are eagerly awaiting the start of “March Madness” and its “bracketology” for predicting all the winners, with odds of 1-in-9.2 quintillion—that’s nine followed by 18 zeros—and is why Warren Buffet will almost certainly not have to pay out the $1 billion he offered for doing so.

The following short story of how basketball came about is worth a quick read before getting to this posting’s DNA sequencing projects, which are not “madness” but definitely long-shot bets—and criticized by some. 

The original 1891 "Basket Ball" court in Springfield College used a peach basket attached to the wall (taken from Wikipedia).

The original 1891 “Basket Ball” court in Springfield College used a peach basket attached to the wall (taken from Wikipedia).

James Naismith (1861 – 1939) was a Canadian American sports coach and innovator. He invented the sport of basketball in 1891 and wrote the original basketball rulebook. At Springfield College, Naismith struggled with a rowdy class which was confined to indoor games throughout the harsh New England winter and thus was perpetually short-tempered. Under orders from Dr. Luther Gulick, head of Springfield College Physical Education, Naismith was given 14 days to create an indoor game that would provide an “athletic distraction.” Gulick demanded that it would not take up much room, could help its track athletes to keep in shape and explicitly emphasized to “make it fair for all players and not too rough.” Naismith did so using the actual “basket and ball” pictured below.

SNPs and GWAS assist in finding the roots of intelligence

Many studies indicate that intelligence is heritable, but to what extent is yet uncertain (taken from the Wall Street Journal via Bing Images).

Many studies indicate that intelligence is heritable, but to what extent is yet uncertain (taken from the Wall Street Journal via Bing Images).

Many of you are well aware of—if not actually involved in—the use of DNA sequence analysis to identify common single nucleotide polymorphisms (SNPs) that are associated with diseases or traits in a study population, relative to a normal control population. Examples of these genome-wide association studies (GWAS) included and were principally enabled in the 1990s by high-density “SNP chips” developed by Affymetrix and then Agilent. While technically straightforward, there’s a lot of genetics and not-so-simple statistics to deal with in designing GWAS and—especially—properly interpreting the results.

In the future, Junior’s DNA sequence could implicate other reasons for his failing academic performance, e.g. not studying enough (taken from dailymail.co.uk via Bing Images).

In the future, Junior’s DNA sequence could implicate other reasons for his failing academic performance, e.g. not studying enough (taken from dailymail.co.uk via Bing Images).

Now, following the advent of massively parallel “next generation” sequencing (NGS) platforms from Illumina and Life Technologies, whole genomes of larger populations (i.e. “many” 1,000s of individuals) can be studied, and less common (aka rare) SNPs can be sought. All of this has fueled pursuit of more challenging—and controversial—GWAS.

So it is the following two ongoing stories that I’ve referred to as the search for genius genes. One conceived by Bowen Zhao—a teenaged Chinese high-school dropout—aiming to find the roots of intelligence in our DNA by sequencing the “off-the-chart” super-smarties, and a newer project by Jonathan Rothberg—über-famous founder of Ion Torrent, which commercialized the game-changing semiconductor-sequencing technology acquired for mega millions by Life Tech—aimed at identifying the roots of mathematical ability by, need I say, Ion Torrent sequencing.

From Chinese high-school dropout to founder of a Cognitive Genomics Unit

It’s a gross understatement to say that Mr. Bowen Zhao is an interesting person—he’s actually an amazing person. As a 13 year old in 2007, he skipped afternoon classes at his school in Beijing and managed to get an internship at the Chinese Academy of Agricultural Sciences where he cleaned test tubes and did other simple jobs. In return, the graduate students let him borrow genetics textbooks and participate in experiments, including the sequencing of the cucumber genome. When the study of the cucumber genome was published in Nature Genetics in 2009, Mr. Zhao was listed as a co-author at the age of 15.

Tantalized by genomics, Mr. Zhao quit school and began to work full-time at BGI Shenzhen (near Hong Kong), one of the largest genomics research centers in the world. BGI (formerly known as the Beijing Genomics Institute) is a private company—partly funded by the Chinese government—that significantly expanded its sequencing throughput last year by acquiring Complete Genomics of Mountain View, California.

Mr. Bowen Zhao is a young researcher with amazing accomplishments (taken from thetimes.co.uk via Bing Images)

Mr. Bowen Zhao is a young researcher with amazing accomplishments (taken from thetimes.co.uk via Bing Images)

The BGI project is sequencing DNA from IQ outliers comparable to Einstein (taken from rosemaryschool.org via Bing Images).

The BGI project is sequencing DNA from IQ outliers comparable to Einstein (taken from rosemaryschool.org via Bing Images).

In 2010, BGI founded the Cognitive Genomics Unit and named Mr. Zhao as its Director of Bioinformatics. The Cognitive Genomics Unit seeks to better understand human cognition with the goal of identifying the genes that influence intelligence. Mr. Zhao and his team are currently using more than 100 state-of-the-art next generation sequencers to decipher some 2,200 DNA samples from some of brightest people in the world—extreme IQ outliers. The majority of the DNA samples come from people with IQs of 160 or higher, which puts them at the same level as Einstein. By comparison, average IQ in any population is set at 100, and the average Nobel laureate registers at around 145. Only one in every 30,000 people (0.003%) would qualify to participate in the BGI project.

In an article by Gautam Naik of the Wall Street Journal, Mr. Zhao is quoted as saying that “people have chosen to ignore the genetics of intelligence for a long time.” Mr. Zhao, who hopes to publish his team’s initial findings this year, added that “people believe it’s a controversial topic, especially in the West [but] that’s not the case in China,” where IQ studies are regarded more as a scientific challenge and therefore are easier to fund.

According to Naik, the roots of intelligence are a mystery, and studies show that at least half of IQ variation is inherited. While scientists have identified some genes that can significantly lower IQ—in people afflicted with mental retardation, for example—truly important genes that affect normal IQ variation have yet to be pinned down.

The BGI researchers hope to crack the problem by comparing the genomes of super-high-IQ individuals with the genomes of people drawn from the general population. By studying the variation in the two groups, they hope to isolate some of the hereditary factors behind IQ. Their conclusions could lay the groundwork for a genetic test to predict a person’s inherited cognitive ability. Although such a tool could be useful, it also might be divisive.

“If you can identify kids who are going to have trouble learning, you can intervene” early on in their lives, through special schooling or other programs, says Robert Plomin, Professor of Behavioral Genetics at King’s College, London, who is involved in the BGI project and quoted by Naik.

Critics, however, worry that genetic data related to IQ could easily be misconstrued—or misused. Research into the science of intelligence has been used in the past “to target particular racial groups or individuals and delegitimize them,” said Jeremy Gruber, President of the Council for Responsible Genetics, a watchdog group based in Cambridge, Massachusetts. “I’d be very concerned that the reductionist and deterministic trends that still are very much present in the world of genetics would come to the fore in a project like this,” Gruber added.

Obtaining access to ‘genius genes’ wasn’t easy

Getting DNA to sequence from super-smart people was easier said than done. According to Naik, Zhao’s first foray into the genetics of intelligence was a plan to collect DNA from high-achieving kids at local high schools. It didn’t work. “Parents were afraid [of giving consent] because their children’s blood would be taken,” Zhao told Naik.

In the spring of 2010, Stephen Hsu—a theoretical physicist from the University of Oregon (now at Michigan State University) who was also interested in the genetics of cognitive ability—visited BGI and joined Zhao to launch the BGI intelligence project. One part of the plan called for shifting to saliva-based DNA samples obtained from mathematically gifted people, including Chinese who had participated in mathematics or science Olympiad training camps. Another involved the collection of DNA samples from high-IQ individuals from the U.S. and other countries, including those with extremely high SAT scores, and those with a doctorate in physics or math from an elite university. In addition, anyone could enroll via BGI’s website—if they met the criteria—as have about 500 qualifying volunteers to date.

Interestingly, most of the samples so far have come from outside of China. The main source is Prof. Plomin of King’s College, who for his own research had collected DNA samples from about 1,600 individuals whose IQs were off the charts. Those samples were obtained through a U.S. project known as the Study of Mathematically Precocious Youth, now in its fourth decade. Dr. Plomin tracked down 1,600 adults who had enrolled as kids in the U.S. project, now based at Vanderbilt University. Their DNA contributions make up the bulk of the BGI samples.

Frequently asked questions about the BGI intelligence project, as well as a link to the detailed project proposal, can be read by clicking here. The penultimate and last paragraphs of the introductory section of this proposal are the following:

The brain evolved to deal with a complex, information-rich environment. The blueprint of the brain is contained in our DNA, although brain development is a complicated process in which interactions with the environment play an important role. Nevertheless, in almost all cases a significant portion of cognitive or behavioral variability in humans is found to be heritable—i.e., attributable to genetic causes.

The goal of the BGI Cognitive Genomics Lab (CGL) is to investigate the genetic architecture of human cognition: the genomic locations, allele frequencies, and average effects of the precise DNA variants affecting variability in perceptual and cognitive processes. This document outlines the CGL’s proposal to investigate one trait in particular: general intelligence or general mental ability, often referred to a “g.”

On Jan 1st 2014, I contacted Prof. Hsu, who coauthored BGI’s “g” proposal, and asked him to clarify whether genome sequencing was in fact being used, as opposed to SNP genotyping chips that were specified in the aforementioned proposal’s Materials and Methods section. I also inquired as to whether any results have been published. His reply on the same day was that the “initial plan was SNPs but [was] upgraded to sequencing. No results yet.”

Stay tuned.

Jonathan Rothberg’s ‘Project Einstein’ taps 400 top mathematicians

In the October 31st 2013 issue of Nature, Erika Check Hayden reported on ‘Project Einstein,’ Ion Torrent founder/inventor/serial entrepreneur Jonathan Rothberg’s new venture aimed at identifying the genetic roots of math genius.

Jonathan Rothberg, founder of CuraGen, 454 Life Sciences, Ion Torrent, Rothberg Center for Childhood diseases, and RainDance Technologies (taken from nathanielwelch.com via Bing Images).

Jonathan Rothberg, founder of CuraGen, 454 Life Sciences, Ion Torrent, Rothberg Center for Childhood diseases, and RainDance Technologies (taken from nathanielwelch.com via Bing Images).

According to Check Hayden’s news article, Rothberg and physicist/author Max Tegmark at MIT in Cambridge, “will be wading into a field fraught with controversy” by enrolling about 400 mathematicians and theoretical physicists from top-ranked US universities in ‘Project Einstein’ to sequence the participants genomes using Ion Torrent machines that Rothberg developed. Critics claim that the study population, like BGI’s “g” project, is too small to yield meaningful results for such complex traits. Check Hayden adds that “some are concerned about ethical issues. If the projects find genetic markers for math ability, these could be used as a basis for the selective abortion of fetuses or in choosing between embryos created through in vitro fertilization.” She says that Rothberg is pushing ahead, and quotes him as stating, “I’m not at all concerned about the critics.”

On the positive side, Prof. Plomin mentioned above in BGI project “g” is said to believe that there is no reason why genome sequencing won’t work for math ability. To support this position, Plomin refers to his 2013 publication entitled Literacy and numeracy are more heritable than intelligence in primary school, which indicates that as much as two-thirds of a child’s mathematical aptitude seems to be influenced by genes.

I’ll be keeping tabs on the project to see how it progresses and how the ethics issue plays out.

Genetics of intelligence is complex and has foiled attempts at deciphering

After reading about the scientifically controversial aspects of both project “g” and ‘Project Einstein,’ I became curious about the outcomes of previous attempts to decipher the genetic basis of intelligence. There was way too much literature to delve into deeply, but a 2013 New Scientist article by Debora MacKenzie entitled ‘Intelligence genes’ evade detection in largest study is worth paraphrasing, as it distills out some simplified takeaways from the referenced study by Koellinger and 200 (!) collaborators published in Science.

  • This team of researchers assembled 54 sets of data on more than 126,000 people who had their genomes analyzed for 2.5 million common SNPs, and for whom information was available on length and level of education. Study organizer Koellinger admits that educational achievement is only a rough proxy for intelligence, but this information was available for the requisite large number of people.
  • Three SNPs from 100,000 people correlated significantly with educational achievement, and were tested against SNPs from the other 26,000 people. The same correlations held, replicating the first analysis. However, the strength of the correlations for each SNP accounted for at most 0.002% of the total variation in educational attainment.
  • “Probably thousands of SNPs are involved, each with an effect so small we need a much larger sample to see it,” says Koellinger. Either that, or intelligence is affected to a greater degree than other heritable traits by genetic variations beyond these SNPs—perhaps rare mutations or interactions between genes.
  • Robert Plomin adds that whole genome sequencing, as being done by BGI, allows researchers to “look for sequence variations of every kind.” Then, the missing genes for intelligence may finally be found, concludes MacKenzie.

Parting Thoughts 

Most, if not all of you, will agree with the contention that a human being is not merely a slave to his or her genes. After all, hasn’t determinism been swept away by the broom of quantum mechanical probabilities as a physical basis of free will? If so, then what role does inherited genetics actually play in intelligence? While the answer to this rhetorical question is obviously not simple, and still hotly debated, I found my thoughts to be largely reflected by a posting at Rose Mary School paraphrased as follows, keeping in mind that all analogies are imperfect:

Human life has been compared to a game of cards. At birth, every person is dealt a hand of cards—i.e., his or her genetic make-up. Some receive a good hand, others a less good one. Success in any game, however, is almost always a matter of education, learning, and culture. For sure, there are often certain innate qualities that will give one person an advantage over another in a specific game. However, without having learned the game and without regular and rigorous practice, nobody will ever become a champion at any game. In the same way the outcome of the game of life is not solely determined by the quality of a person’s initial hand of cards, but also by the way in which he or she takes part in the game of life. His or hers ability to take part in the game of life satisfactorily, perhaps even successfully, will be determined to a very large extent by the quality and quantity of education that he or she has enjoyed.

When I gave advice to students, as a teacher, it was very simple and what I did—and still do—myself: “study hard, work harder, and success will follow.”

As always, your comments are welcomed.

DNA Barcoding Exposes Scary Data for Herbal Products

  • Americans spend $5 billion annually for products with unproven benefits
  • Recent studies show majority of herbal products are contaminated or inauthentic
  • Dietary supplements account for ~20% of drug-related liver injuries
  • What can be done to protect consumers?

In August 1873, Darwin had ‘a fit’, in which he temporarily lost his memory and could not move. He recovered, and busied himself in work on the cross- and self-fertilization of plants. Published in 1876, Effects of Cross and Self Fertilisation in the Vegetable Kingdom provided ample proof of his belief that cross-fertilized plants produced vigorous off-spring that were better adapted to survive. Taken from otago.ac.nz via Bing Images

Since this post comes on the heels of Charles Darwin Day (February 12th ), it’s apropos to recognize Darwin’s plant-related scientific contributions dealing with plant movement and cross-fertilization—largely as the result of his keen observations and powerful reasoning that we should strive to emulate in our scientific pursuits. Darwin’s sense of scientific integrity seems to have been lost on the world of herbal products according to several recent studies and reports. It appears this industry is in need of some policing and compliance that may well come from DNA barcoding.   

According to Anahad O’Connor’s sobering article in the NY Times, Americans spend $5 billion a year on unproven herbal supplements—promising everything from fighting colds to boosting memory. Given the size of this market, it’s alarming that these products are oftentimes contaminated and/or mislabeled. Hmmm, sound familiar?  You may be recalling one of my posts from last year that explored analogous DNA-based findings for meat and fish products indicating global gross negligence or fraud. To quote Yogi Berra, “it’s like déjà vu, all over again”. Before delving into the details of this unsettling—if not downright scary—situation, let’s briefly discuss some background concepts that will enable us to understand how DNA barcoding may help bring honesty and transparency to the herbal products industry.

What is DNA Barcoding?

Since the herbal products exposé described below involves use of DNA barcoding, some readers may want to know what this methodology involves. Fortunately, Cold Spring Harbor Laboratory provides a website called DNA Barcoding 101 that provides the following snippets of information, as well as detailed “how to” protocols in a downloadable PDF.

Taxonomy is the science of classifying living things according to shared features. Less than two million of the estimated 5-50 million plant and animal species have been identified. Scientists agree that the yearly rate of extinction has increased from about one species per million to 100-1,000 per million. This means that thousands of plants and animals are lost each year, and most of these have not yet been identified.

Classical taxonomy falls short in this race to catalog biological diversity before it disappears. Specimens must be carefully collected and handled to preserve their distinguishing features. Differentiating subtle anatomical differences between closely related species requires the subjective judgment of a highly trained specialist—and few are being produced in colleges today.

Now, DNA barcodes allow non-experts to objectively identify species—even from small, damaged, or industrially processed material. Just as the unique pattern of bars in a universal product code (UPC) identifies each consumer product, a “DNA barcode” is a unique pattern of DNA sequence that identifies each living thing. DNA barcodes, about 700 nucleotides in length, can be quickly processed from thousands of specimens and unambiguously analyzed by computer programs. Barcoding relies on short, highly variably regions of the genome. With thousands of copies per cell, mitochondrial and chloroplast sequences are readily amplified by PCR, even from very small or degraded specimens, to enable DNA sequencing of the barcode.

Herbal product DNA barcodes are, in principle, similar to those pictured below that exemplify unique characterization of two different cryptic species of a butterfly, which appear visually to be nearly identical, and two genera of owl that are visually distinct.

Four-color DNA sequencing trace showing sequence of T (red), C (blue), G (black), and A (green) bases that comprises a barcode and can be redrawn as depicted below (taken from srmgenetics.info via Bing Images).

Four-color DNA sequencing trace showing sequence of T (red), C (blue), G (black), and A (green) bases that comprises a barcode and can be redrawn as depicted below (taken from srmgenetics.info via Bing Images).

DNA barcodes identify all living things (taken from boomersinfokiosk.blogspot.com via Bing Images).

DNA barcodes identify all living things (taken from boomersinfokiosk.blogspot.com via Bing Images).

Selling Herbal Products is BIG Business…But Who’s Watching Out for You?

The international trade in herbal products is a major force in the global economy and the demand is increasing in both developing and developed nations. According to a recent report, there are currently more than 1,000 companies producing medicinal plant products with annual revenues in excess of $60 billion. Notably, medicinal herbs now constitute the most rapidly growing segment of the North American alternative medicine market, with over 29,000 herbal substances generating billions of dollars in trade. For those of you who may be interested, I found this list of the top 10 best-selling herbal supplements in the US, among which are Ginseng (#9), Purple coneflower (#6), Ginkgo (#4), Cranberry (#2), and—surprising to me—Soy (#1).

Ginseng berry growth is restricted to certain areas of the world due to climate and weather conditions, making it difficult to cultivate. When this rare berry is picked, it is after a three- to four-year wait, and then the harvest must occur during a short, two-week period. Ginseng berry contains potent antioxidants called ginsenosides—such as Ginsenoside Rg1 pictured here—that are more abundant and different than those found in the root of the ginseng plant. These nutrients are believed to enhance the body's natural defense system to aid against invading free radicals (taken from eexcel.net via Bing Images and Wikipedia.org).

Ginseng berry growth is restricted to certain areas of the world due to climate and weather conditions, making it difficult to cultivate. When this rare berry is picked, it is after a three- to four-year wait, and then the harvest must occur during a short, two-week period. Ginseng berry contains potent antioxidants called ginsenosides—such as Ginsenoside Rg1 pictured here—that are more abundant and different than those found in the root of the ginseng plant. These nutrients are believed to enhance the body’s natural defense system to aid against invading free radicals (taken from eexcel.net via Bing Images and Wikipedia.org).

Although soy has been a staple of Asian cuisine for centuries, Westerners have only recently become aware of its valuable health benefits. Soy is a source of easily digestible protein and contains no saturated fat or cholesterol. Scientists have demonstrated through over 40 studies, spanning a 20-year period that eating 25 grams of soy per day helps reduce the risk of America's number one killer—heart disease. According to the US Food and Drug Administration (FDA), eating 25 grams of soy per day as a part of a low fat, low-cholesterol diet may reduce the risk of coronary heart disease (taken from eexcel.net via Bing Images).

Although soy has been a staple of Asian cuisine for centuries, Westerners have only recently become aware of its valuable health benefits. Soy is a source of easily digestible protein and contains no saturated fat or cholesterol. Scientists have demonstrated through over 40 studies, spanning a 20-year period that eating 25 grams of soy per day helps reduce the risk of America’s number one killer—heart disease. According to the US Food and Drug Administration (FDA), eating 25 grams of soy per day as a part of a low fat, low-cholesterol diet may reduce the risk of coronary heart disease (taken from eexcel.net via Bing Images).

Unfortunately, product adulteration and ingredient substitution is not uncommon in the medicinal herb and dietary supplement markets, as species of inferior quality are often substituted for those of a higher value. This practice constitutes not only product fraud, but according to the World Health Organization (WHO), it is a serious threat to consumer safety, as commented on below.

Currently, there are no best practices in place for identifying the species of the various ingredients used in herbal products. This is because the diagnostic morphological features of the plants cannot typically be assessed from powdered or otherwise processed biomaterials. As a result, the marketplace is prone to contamination and possible product substitution, which dilute the effectiveness of otherwise potentially useful remedies. Fortunately, DNA barcoding can now be used to combat this serious situation.

Report Reveals Rampant Contamination and Substitution

Dr. Steven G. Newmaster, Associate Professor, Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada (taken from uoguelph.ca).

Dr. Steven G. Newmaster, Assoc. Prof., Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Ontario, Canada (taken from uoguelph.ca).

In October of 2013, a team of Canadian and Indian scientists led by Dr. Steven G. Newmaster published in BMC Medicine a report entitled DNA barcoding detects contamination and substitution in North American herbal products that has received widespread media attention. This study utilized blind sampling of commercially available herbal products, which were tested for authentication of plant ingredients using a Standard Reference Material (SRM) herbal DNA barcode library. The research questions focused on the following three areas. (1) Authentication: is the herbal species on the label found in the product? (2) Substitution: is the main herbal ingredient substituted by other species? (3) Fillers: are any unlabeled fillers used?

They tested the authenticity of 44 herbal products (41 capsules; 2 powders; 1 tablet) representing 12 companies. The samples were collected in the greater Toronto area in Canada, with several samples mailed from distributors in the US. All products are available to consumers in both Canada and the US. The herbal product samples represented 30 herbal species that were each represented by 2 or 3 different companies. The samples were submitted in a blind test for authentication (product no. label only) using PCR-BigDye® Sanger sequencing-based DNA barcoding at the Centre for Biodiversity Genomics within the Biodiversity Institute of Ontario, University of Guelph.

The following are some of their sobering findings:

  • Only 2 of the 12 companies tested provided authentic products without substitutions, contaminants or fillers.
  • Nearly 60% of the herbal products contained plant species not listed on the label.
  • Product substitution was detected in 32% of the samples.
  • More than 20% of the products included fillers such as rice, soybeans and wheat not listed on the label.

This graphical summary of the results taken from the NY Times speaks for itself.

1105-HERBAL-FOR-FRONT-popup

In a follow-up press release from the University of Guelf, Dr. Newmaster said that “[c]ontamination and substitution in herbal products present considerable health risks for consumers.” He added that “[w]e found contamination in several products with plants that have known toxicity, side effects and/or negatively interact with other herbs, supplements and medications.” The statement added that one product labeled as St. John’s Wort contained Senna alexandrina, a plant with laxative properties—not intended for prolonged use, as it can cause chronic diarrhea and liver damage, and negatively interacts with immune cells in the colon. Also, several herbal products contained Parthenium hysterophorus (feverfew), which can cause swelling and numbness in the mouth, oral ulcers and nausea. It also reacts with medications metabolized by the liver.

Furthermore, one ginkgo product was contaminated with Juglans nigra (black walnut), which could endanger people with nut allergies. Unlabeled fillers such as wheat, soybeans and rice are also a concern for people with allergies or who are seeking gluten-free products, Newmaster said. “It’s common practice in natural products to use fillers such as these, which are mixed with the active ingredients. But a consumer has a right to see all of the plant species used in producing a natural product on the list of ingredients.”

Dietary Supplements Account for ~20% of Drug-related Liver Injuries

Consumer safety is not a theoretical concern. According to Anahad O’Connor’s follow-on and lengthier Dec 22nd 2013 NYTimes article entitled Spike in Harm to Liver is Tied to Dietary Aids, “[d]ietary supplements account for nearly 20 percent of drug-related liver injuries that turn up in hospitals, up from 7 percent a decade ago, [that] according to an analysis by a national network of liver specialists. The research included only the most severe cases of liver damage referred to a representative group of hospitals around the country, and the investigators said they were undercounting the actual number of cases.” The article features a 17 year-old male who suffered severe liver damage after using a concentrated green tea extract he bought at a nutrition store as a “fat burning” supplement. The damage was so extensive that he was put on the waiting list for a liver transplant. Fortunately he recovered and the transplant wasn’t necessary.

This NYTimes article is well worth reading, and provided further evidence that tighter regulations are needed.

Conclusions

Given the sobering significance of the above study by Newmaster and coworkers, I thought it was better to directly quote concluding remarks by these authors, rather than selectively paraphrase and oversimplify the situation:

“Currently there are no standards for authentication of herbal products. Although there is considerable evidence of the health benefits of herbal medicine, the industry suffers from unethical activities by some of the manufacturers, which includes false advertising, product substitution, contamination and use of fillers. This practice constitutes not only food fraud, but according to the WHO, serious health risks for consumers. A study of health claims made by herbal product manufacturers on the internet found that 55% of manufacturers illegally claimed to treat, prevent, diagnose or cure specific diseases. Regulators such as the FDA and Canadian Food Inspection Agency (CFIA) may not have the resources to adequately monitor the dietary supplement manufacturers and their advertising claims, and there are concerns that the current regulatory system is not effective in protecting consumers from the risks associated with certain herbal products. Chemical research studies have documented poor quality control and high content variability of active ingredients among products from a variety of manufacturers of herbal supplements. This is partly because herbs contain complicated mixtures of organic chemicals, the levels of which may vary substantially depending upon many factors related to the growth, production and processing of each specific herbal product. Although many manufacturers provide products with consistent levels of active ingredients through a process known as chemical standardization, this technique has uncertain effects on the safety and efficacy of the final product.”

“Many of the dangers of commercial plant medicine have been brought to light by DNA technology based studies that have identified contamination of herbal products with poisonous plants. Eroding consumer confidence is driving the demand for a product authentication service that utilizes molecular biotechnology. One approach to vetting herbal product substitution and contamination is product authentication using DNA barcoding. Research studies such as ours and others reinforce the importance of using DNA barcoding in the authentication of socioeconomically important herbal species. We suggest that the herbal industry should voluntarily embrace DNA barcoding for authenticating herbal products through testing of raw materials used in manufacturing products, which would support sovereign business interests and protect consumers. This would be a minor cost to industry with a limited amount of bulk product testing, which would certify a high quality, authentic product. If the herb is known to have health benefits and it is in the product, then this would provide a measure of quality assurance in addition to consistent levels of active ingredients. Currently we are building an SRM DNA barcode library for commercial herbal species and standard testing procedures that could be integrated into cost effective ‘best practices’ in the manufacturing of herbal products. This would provide industry with a competitive advantage as they could advertise that they produce an authentic, high quality product, which has been tested using DNA-based species identification biotechnology, therefore gaining consumer confidence and preference. This approach would support the need to address considerable health risks to consumers who expect to have access to high quality herbal products that promote good health.”

I for one would gladly pay a premium for herbal products that are “DNA Barcode Certified”.

Wouldn’t you?

As always, your comments are welcomed.

Postscript

After I finished writing this post, I went back to the aforementioned BMC Medicine article by Newmaster and coworkers to use its Google-based link to blogs citing this publication. While most were recaps of the report, one entitled It’s Now Herbal Products’ Turn to (Unfairly) Wear the Scarlet Letter caught my attention. I won’t provide a synopsis here, but you may want to read the blogger’s rather different, über critical commentary that seems (unfairly) biased to me.

If you’re interested in all facets of DNA barcoding, I highly recommend visiting the DNA Barcoding blog by Dirk Steinke at Guelf University that has new posts daily.

For those of you who are educators, the BioBelize website provides an example of how secondary education programs in New York City and Belize came together to define and implement DNA Barcoding curricula to encourage young student interest and involvement in this aspect of science.

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.

Sequencing DNA on Mars – Science Far Out or Far Out Science?

There’s no shortage of clever “lab-on-chip” (LOC) devices for all sorts of interesting point-of-use applications; however, investigating the feasibility of an LOC for sequencing DNA on Mars seemed “far out” to me both extraterrestrially and scientifically. After some poking around in the literature to learn more about this research – which turns out not to be too far-fetched – I thought it would be worth sharing here some background about astrobiology and some experimental specifics.

What are Astrobiology and NASA’s Astrobiology Program?

According to the US National Aeronautics and Space Administration (NASA), “astrobiology is the study of the origin, evolution, distribution, and future of life in the universe. This multidisciplinary field encompasses the search for habitable environments in our Solar System and habitable planets outside our Solar System, the search for evidence of prebiotic chemistry and life on Mars and other bodies in our Solar System, laboratory and field research into the origins and early evolution of life on Earth, and studies of the potential for life to adapt to challenges on Earth and in space.”

Future astrobiologist in search of a sample for possible DNA detection? (Bing Images)

Future astrobiologist in search of a sample for possible DNA detection? (Bing Images)

NASA’s Astrobiology Program addresses three fundamental questions:

  • How does life begin and evolve?
  • Is there life beyond Earth and, if so, how can we detect it?
  • What is the future of life on Earth and in the universe?

Needless to say, these are truly profound questions requiring very long-term commitments by interdisciplinary teams supported by huge – dare I say astronomical – budgets. Readers interested in perusing a presentation detailing how NASA’s entire 2014 budget request of ~$18 billion will be spent can click here, while information about NASA funding opportunities can be found by clicking here. Incidentally, NIH’s budget request for FY 2014 is ~$31 billion.

Primordial Soup

Oparin

Alexander Ivanovich Oparin graduated from the Moscow State University in 1917 and became a Professor of Biochemistry there in 1927. Many of his early papers were on plant enzymes and their role in metabolism. In 1924 he put forward a theory of life on Earth developing through gradual chemical evolution of carbon-based molecules in primordial soup.

Theories and research on how life – as we know it – began and evolved has received considerable attention since 1924 when Soviet biologist Alexander Oparin proposed a theory of the origin of life on Earth through gradual chemical evolution of molecules that contain carbon in the “primordial soup.”

The research continued with Stanley L. Miller’s, now classic paper, “A Production of Amino Acids Under Possible Primitive Earth Conditions” in Science in 1953 – the same year as Watson & Crick’s discovery of the structure of DNA. Miller’s work involved paper chromatography after electrical discharges in a mixture of CH4, NH3, H2O and H2. Extensions of this line of experimentation to detect nucleobases formed under possible prebiotic conditions on Earth that led to evolution are now numerous (click here for more examples).

Possible contamination with terrestrial molecules has been a concern for detection of nucleobases in carbon-rich meteors. However, recent identification of nucleobase analogs 2,6-diaminopurine and 6,8-diaminopurine has been reported by Dworkin and coworkers to strongly support extraterrestrial origin. These investigators conclude that “[b]ecause meteorites may have provided a significant source of prebiotic organic material including purines, it is possible that alternative nucleobases such as 2,6-diaminopurine, 6,8-diaminopurine, xanthine, and hypoxanthine were available for constructing the first genetic molecules” involving an “expanded genetic alphabet,” as proposed in RNA World or all-purine primitive RNA.

According to a NY Times report on September 12th of this year, Dr. Steven Benner – founder of the Foundation for Applied Molecular Evolution – suggested that, if life started out as RNA, minerals containing borate could have been involved in the chemistry of its formation. Since boron has been recently found in a Martian meteorite, Mars might have been a favorable place for RNA to emerge, and for life to start. The report goes on to say that a giant impact could then have kicked up microbe-laden rocks, which latter fell to earth. I should note here that, while this Mars-to-Earth scenario is opposite an Earth-to-Mars pathway discussed below, these “interplanetary nucleic acid-exchange schemes” are, in principle, not mutually exclusive.

Incentive to continue work in this area has been provided in the form of The Origin-of-Life Prize® that is intended to “encourage the pursuit of natural-process explanations and mechanisms of initial ‘gene’ emergence within nature.” Interestingly, this one-time prize worth $1,000,000 that will be paid to the winner(s) as a twenty-year annuity of $50,000 per year “in hopes of discouraging theorists’ immediate retirement from productive careers.”

What is the Possible Origin of Martian Genomes?

One answer to this question is provided by the “common ancestry hypothesis” that involves natural transfer of viable microbes in space, such as from Earth to Mars and Mars to Earth. An international team of scientists, including some from NASA, have published supporting data for the possibility and probability of such transfer of microbes from traveling in meteoroids billions of years ago following the Big Bang development of the universe. Factors taken into account include radiation protection, vacuum, temperatures, acceleration pressures, and spontaneous DNA decay. They conclude that “if microbes existed or exist on Mars, viable transfer to Earth is not only possible but also highly probable, due to microbes’ impressive resistance to the dangers of space transfer and to the dense traffic of billions of Martian meteorites which have fallen on Earth since the dawn of our planetary system. Earth-to-Mars transfer is also possible but at a much lower frequency.” I encourage those of you interested in more information about this possibility, (depicted below), to click here to read Scientific Logic for Life on Mars by Gilbert V. Levin, who was the Principal Investigator of the 1976 Viking mission Mars Lander Labeled Release (LR) experiment.

According to a 2012 article in National Geographic, the LR experiment involved scooping up Martian soil and mixing it with a drop of water containing nutrients from a source of radioactive carbon. The hypothesis was, if the soil contained microbes, metabolism of the nutrients would release either radioactive CO2 or CH4 that could be measured by a radiation detector in the probe. A number of control experiments were also performed. The LR experiment came out positive for life, and the control experiments came out negative.  Unfortunately, two other experiments did not confirm these results and NASA dismissed the possibility of life having been detected. Levin and others have reanalyzed the results and stuck by the claim.

Taken from “Scientific Logic for Life on Mars” by Gilbert V. Levin at www.gillevin.com via Bing Images.

Taken from “Scientific Logic for Life on Mars” by Gilbert V. Levin at www.gillevin.com via Bing Images.

Developing the Search for Extra-Terrestrial Genomes (SETG) Instruments

Feasibility studies aimed at development of instrumentation for SETG have been published in a series of reports by a team of researchers including Christopher E. Carr [MIT & Massachusetts General Hospital (MGH)], Holli Rowedder (MGH), Clarissa S. Lui (MIT), Maria T. Zuber (MIT), and Gary Ruvkun (MGH & Harvard Medical School) – click here for photos and bios of the SETG Team.

In a 2011 report, they addressed various aspects of isolating, extracting, and sequencing nucleic acids in situ on Mars – particularly 16S and 23S ribosomal RNA genes. These genes are used for phylogenic analysis to address whether sequences found on Mars are similar to those on Earth and likely contamination, or indicative of extant Martian life isolated from that on Earth for the past 3+ billion years.

These investigators note that investigating DNA extraction and amplification from Martian soil of unknown composition is a significant issue; however, “Martian soil simulants” have been developed based on spectral characterization of soild matter on Mars from the Viking and Pathfinder landing sites. Among various LOC-compatible DNA purification and concentration methods, synchronous coefficient of drag alteration (SCODA) was said to be a promising new technique—click here for a poster on SCODA applied to a Martian soil analog presented at the Astrobiology Science Conference 2012. Challenges facing all of the other steps in sample-to-answer for a SETG instrument are addressed in the aforementioned 2011 report, which correctly – in my opinion – emphasizes that library preparation will be especially difficult to solve due to there being numerous steps and reagents.

setg

The current SETG instrument contains a 2 cm x 2 cm microfluidic chip module containing tiny nanoliter wells where the real-time polymerase chain reactions occur. Tiny tubes feed in chemicals and blue light illuminates fluorescent dyes that help identify and analyze DNA molecules (picture and caption taken from MIT website).

In addition to the SETG reports, two follow-on reports have been published in Astrobiology this year. In the first of these, Carr et al. demonstrated that Ion Torrent’s semiconductor proton-sensing, optics-free (“no fluorescence”) sequencing chips survive several analogs of space radiation at doses consistent with a 2-year Mars mission, including protons with solar particle event–distributed energy levels and 1 GeV oxygen and iron ions. There was no measurable impact of irradiation at 1 and 5 Gy doses on either sequencing quality or low-level hardware characteristics. The second report by Carr et al. demonstrated that biological and chemical components, such as polymerase, dNTPs, and fluorescent dye molecules survived several analogs of the radiation expected during a 2-year mission to Mars, including proton (H-1), heavy ion (Fe-56, O-18), and neutron bombardment. Other reagents had reduced performance or failed at higher doses. It was concluded that, overall, the findings suggest it is feasible to utilize space instruments with biological components for mission durations up to several years in environments without large accumulations of charged particles, such as the surface of Mars.

After reading these SEGT-related publications, I contacted Prof. Carr to ask if a SEGT instrument was part of NASA’s Mars 2020 Mission that I had read about in a July 27th blog post by Van Kane on The Planetary Society website. The detailed blog was entitled The Mars 2020 Rover In-Depth, which said that NASA’s next major mission to the Red Planet will store samples for eventual return to the Earth. Prof. Carr’s reply to me was that “[w]e are not currently slated for a mission. The Mars 2020 rover mission is likely to include the best package of instruments possible based on the submitted proposals and the priorities outlined in the recent Science Definition Team report….[T]he strawman set of instruments is somewhat similar to those on Curiosity. But there will almost assuredly be some new instrumentation. Sample return caching certainly would be completely new.”

In closing this post, I welcome comments, especially opinions on whether sequencing DNA on Mars is “science far out” or “far out science”?

Postscript

Doubts about Martian methane: On September 19th the Wall Street Journal reported in an online video that NASA scientists say they cannot find any traces (less than one part per billion) of methane in the thin Martian air, dimming hopes that microbes might lurk under the protective blanket of soil on the cold arid world. Also reported were long-range data to the contrary, and mention of upcoming Martian methane-detection missions by India and the European Space Agency.

Soil samples taken and analyzed from this site have been found to contain Martian water. The left dimension of each scoop is 4 cm (~1.5 in)! Courtesy NASA/JPL-Caltech/MSSS via Bing Images.

Soil samples taken and analyzed from this site have been found to contain Martian water. The left dimension of each scoop is 4 cm (~1.5 in)! Courtesy NASA/JPL-Caltech/MSSS via Bing Images.

Mars has a surprising amount of water! One week later on September 27, Curiosity researcher Laurie Leshin and colleagues told Science Magazine that Mars’ dusty red covering holds about 2% water by weight. This could be a useful resource for future astronauts, they say. “If you think about a cubic foot of this dirt and you just heat it a little bit – a few hundred degrees – you’ll actually get off about two pints of water – like two water bottles you’d take to the gym,” Dr Leshin explained. Aside from drinking or farming, water could also be a source of H2 and O2 for fuel and breathing, respectively.

Do you want a one-way ticket to Mars? Mars One is a non-profit organization that plans to establish a permanent human colony on Mars by 2023. The private spaceflight project is led by Dutch entrepreneur Bas Lansdorp, who announced official plans for the Mars One mission in May 2012. In 2022, four carefully selected applicants will be launched in a Mars-bound spaceflight to become the first residents on Mars. Every step of the crew’s journey will be documented for a reality television program that will broadcast 24/7/365. On August 9th, CNN reported that more than 100,000 people have applied for this one-way trip to Mars, hoping to be chosen to spend the rest of their lives on uncharted territory. Who knows, maybe they will be able to extract Martian soil samples in attempts to detect and sequence DNA.

Coincidentally, it was reported in the September 12th, 2013 issue of Nature that NASA is launching a research program to investigate how human and other tissue reacts to time spent in space. Grant applications for up to $500,000 for a 5-year period will be accepted in Fall of 2014.

Artist rendition of the Mars One colony (taken from oditycentral.com via Bing Images)

Artist rendition of the Mars One colony (taken from oditycentral.com via Bing Images) 

 

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.