Virtual Reality for Graphene Nanopores and Space Station Sequencing 

  • Simulated Sequencing Takes Virtual Reality Way Beyond Games  
  • In Silico Simulations Suggest Possible 99.99% Accuracy for Graphene Nanopores 
  • minION Nanopore Sequencer is Sent to the International Space Station

Prelude

oculusvr

Taken from oculusvr.com

This blog is mostly about an international team of researchers who are using Virtual Reality (VR)—in the form of computational modeling—to simulate a new approach to DNA sequencing using nanopores made out of graphene. While VR is a hot trend in all sorts of so-called immersion media, such as those offered by Oculus (that was acquired by Facebook for $2 billion in 2014), computation-based VR has been used by scientists for simulating molecular interactions for a relatively long time. However, extending molecular simulations to complex (aka many-atom) systems like nanopores and DNA has had to wait for bigger, faster, cheaper computing.

In this blog, I’ll also discuss the recent launch of a commercially available nanopore sequencer for the first ever DNA sequencing in space using a self-landing rocket operated by Space X (co-founded by uber-famous multi-billionaire entrepreneur Elon Musk). It’s hard for me to even imagine what seemingly incongruent mix of topics could be more intriguing than these. As the now trendy saying goes, you can’t make this stuff up. But I digress…

Lift off! Taken from am1070theanswer.com                     Self-landing! Taken from indianexpress .com

Lift off! Taken from am1070theanswer.com    –  Self-landing! Taken from    indianexpress.com

Nanopore Sequencing 

baseFrom an earlier blog you’ll know that I’m a huge fan of tiny nanopores for sequencing, which is a 20+ year old concept, as depicted below from a seminal patent wherein DNA was envisaged as moving through a pore-in-lipid bilayer leading to base-dependent transient blockage of ionic current from which sequence is determined.

Taken from nature.com

Taken from nature.com

After two decades, this prophetic concept of nanopore sequencing has recently been realized, and commercialized by Oxford Nanopore Technologies (ONT) using “bionanopore” technology. Comparing the images above and below, you’ll see that bionanopores are, in many respects, quite similar to the first described nanopores, wherein a pore-forming protein, α-hemolysin (gray), is embedded in a lipid bilayer (blue). On the other hand, there is an attached DNA-processive enzyme, 29 DNA polymerase (brown), that feeds in the single strand of DNA for sequencing; details may be found elsewhere.

An alternative strategy for nanopore sequencing is to replace this type of bionanopore (composed of biological macromolecules) with a pore constructed of non-biological materials, notably silicon-based semiconductors that enable electrical signal generation and data processing. This would-be evolution of nanopore sequencing from biological constructs to various types of solid-state materials can be read about elsewhere.

Whither Goest Graphene?

It seems the next step in the progression of nanopore technology is those made of graphene—the trivial name for a very special form of carbon that was long known but exceedingly difficult to make. In fact, the process is so difficult that Andre Geim and Kostya Novoselov at The University of Manchester were awarded the 2010 Nobel Prize in Physics for their work enabling the production and characterization of graphene.

Geim and Novoselov. Taken from rsc.org 

Geim and Novoselov. Taken from rsc.org

Graphene is a two-dimensional array or “sheet” of carbon atoms that is usually depicted by the ball-and-stick model (pictured at the left below) as a one-atom-thick sheet of otherwise infinite dimensions. Since nothing is infinite in the real world, sheets of graphene have edges to which hydrogen is bonded, but for simplicity is ignored. This carbon-carbon bonding with carbon-hydrogen edges is akin to that in polycyclic aromatic hydrocarbons familiar to readers who are chemists.

Taken from 3dprint.com  

Taken from 3dprint.com

Taken from Bayley (2010) in nature.com

Taken from Bayley (2010) in nature.com

Because of graphene’s unique electrical properties and single-atom-thin structure, the basic idea is that a nanometer-size hole in graphene might be made—somehow—to allow DNA and ions to pass through and thus generate electrical signals—somehow—that are accurately deciphered—somehow—into DNA sequence. Oh, and let’s not forget that this sequence information must differentiate—somehow—3’->5’ from 5’->3’ directional pass through. All these “somehows” are meant to indicate that it’s far easier to imagine the concept of graphene nanopore sequencing, as fancifully shown below, than to actually do it.

Taken from Mechant et al. Nano Letters (2010)

Taken from Mechant et al. Nano Letters (2010)

The most daunting practical problem deals with how to “drill” tiny holes in graphene. One approach has been to use controlled electron-beam exposure in a transmission electron microscope. Initial demonstration of this approach was published in 2010 by Merchant et al. in Nano Letters in a paper titled DNA Translocation through Graphene Nanopores, from which the schematic left is taken.

In this device, a few-atoms-layer piece of graphene (1-5 nm thick) having an ~10 nm hole is suspended over a 1 μm diameter hole in a 40 nm thick silicon nitride (SiN) membrane suspended over an ~50 × 50 μm2 aperture in a silicon chip coated with a 5 μm silicon oxide (SiO2) layer in such a way that a bias voltage (VB) is applied between the reservoirs to drive DNA through the nanopore. Although DNA could be detected, the graphene pore size was too big to allow sequence detection.

Taken from Chang et al. Nano Letters (2010)

Taken from Chang et al. Nano Letters (2010)

Similar studies by Schneider et al. were also reported in Nano Letters in 2010, which appears to be a watershed year for this journal inasmuch as another noteworthy nano-detection scheme for DNA was described therein by Chang et al.—but with an important new feature. Namely, using gold electrodes (in yellow, below) separated by only 2 nm and conjugated to dC, a derivative of dG (blue balls) apparently was able to H-bond (magenta) to dC—based on dC-dG complementarity and detected as electron tunneling signals. This transient, base pair-specific H-bonding is what has now been further investigated by others albeit in the following form of Virtual Reality.

Virtual Reality Nanopore Sequencing

In contrast to the above “real” experiments, others have simulated reality using mathematical calculations based on theoretical chemistry, which is Virtual Reality that has physical significance well beyond simply playing games. Mathematical modeling or computation simulations are phrases generally used to describe these so-called in silico “experiments” that serve as indications of what could be done, in theory, if this Virtual Reality is actually translatable to the real world. But I digress…

An international team of investigators in the U.S., Germany, and Netherlands has recently reported studies titled Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing. Although this article is a dreaded “pay-to-read” article, there is a brief news piece about it at the website for the U.S. National Institute of Standards and Technology (NIST) where some of this work was conducted.

Taken from nist.gov

Taken from nist.gov

As is evident from the schematic shown below, these investigators borrowed from the aforementioned types of publications to imagine a graphene nanopore having its internal edges functionalized with nucleobase moieties that could potentially H-bond with DNA bases in a sequence specific manner—à la Chang et al. Under appropriate conditions, this could provide the basis for sequencing via measurement of induced current fluctuations.

More specifically, they imagined a sheet (aka ribbon) of graphene 4.5 x 5.5 nm with several nucleobase moieties attached to a 2.5 nm nanopore. In animated simulations (which are linked at the NIST website), you can watch how this sensing device would perform at room temperature in water with attached cytosine H-bonding to detect G in DNA.

When you watch this simulation, you’ll immediately notice how “wiggly” DNA is due to random motions of its constituent groups and atoms. You’ll also see detection of each translocating (i.e. passing G) as an increasing signal being recorded in real-time. While time as a parameter in this simulation is real, the simulation itself is not real, but rather virtual reality based on state-of-the art theoretical calculations by a computer.

With that caveat in mind, the performance was said to be 90% accurate (due to missed bases rather than wrongly detecting a base) at a rate of 66 million bases per seconds, which to me is mindboggling ultra-fast. Moreover, if this device could be fabricated as four sequentially-located graphene pores each functionalized with either C, G, A, or T, the researchers estimate that “proofreading” would increase accuracy to 99.99%, as required for sequencing the human genome.

Virtual reality is, well, not reality. And sometimes dreams and reality go in opposite directions. However, if indeed the above imaginary device and simulations were to become reality—nanopore sequencing would indeed be advanced dramatically from today’s performance.

ONT’s minION Sequencer in Space

In transitioning back to reality, it’s almost unbelievable to me that ONT’s minION nanopore sequencer—which I’ve blogged about before—was sent to the International Space Station (ISS) in April 2016 to carry out the first ever sequencing of DNA in space. If that wasn’t enough “buzz”, then the fact that this was achieved by uber-famous Elon Musk’s Space X company made it way more so, along with much ado in successfully landing the rocket’s first-stage on a relatively tiny platform in the ocean. This is all amazing stuff.  And to think that not so long ago, we were thinking how great it would be if self-landing rockets were really possible, not just a fun concept in video games and sci-fi movies!  Maybe virtual reality isn’t that far from becoming reality, but I digress….

The first aim of putting the minION nanopore into space is to demonstrate the feasibility of nanopore sequencing in microgravity. That being done would then allow use of the minION to rapidly sequence astronaut samples in the ISS to diagnose, for example, an infectious disease or other health issue.

Interested readers can peruse elsewhere much more about this historic milestone, as well as watch and listen to a short (but very exciting) video titled Space Station Live: Big DNA Science in a Small Package. The video was posted on Twitter on July 21st and features the minION device (aka The Biomolecular Sequencer).

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from spaceref.com

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from spaceref.com

I look forward to learning the results of the minIONs research in space. I hope that it’s “mission accomplished!” and of great use in years to come. BTW, if you’re a “space buff” like me, you can watch and listen to ISS-Mission Control live streaming 24-7 at this website.

As usual, your thoughts about this blog are welcomed as comments.

Sequencing Trifecta for Top 10 Innovations of 2015

  • Sequencing Sweeps The Scientist’s Top 3
  • Diverse Array of Research and Diagnostic Products Round Out Top 10
  • I Predict 3 Winners for 2016. What Are Yours?
Taken from the-scientist.com.

Taken from the-scientist.com.

Welcome to my first blog of the New Year, 2016! There is a trove of topics in my queue of blogs, and I invite you to check them out every other Tuesday throughout the year. As in the past, this first blog of the year comments on the Top 10 Innovations in 2015 that were picked by a panel of judges and published last month in The Scientist. As a side note, you can also peruse TriLink’s top products of 2015 and predictions for 2016 by clicking here.

When you read about these winners, you’ll find out that 1st, 2nd and 3rd place involve sequencing—a trifecta in parimutuel betting on horse races—that were kind of a sure thing (to continue my analogy to betting) based on sequencing products also being in the top spots in the previous year picks. This preeminence of sequencing will likely continue, as I’ll explain at the end of this blog with my win, show and place bets for next year.

Taken from wikipedia.org.

From wikipedia.org.

Continue reading

Nanopore Sequencing: 20 Years On 

  • Once Only a Dream, Nanopore Sequencing is Now Reality
  • Oxford Nanopore Technologies MinION Sequencer Apps Spotlighted  
  • Fully Automated Sample Prep and Higher Throughput Coming Soon
Dream Messenger oil painting by Leszek Andrzej Kostuj with nanopore-like eyes. Taken from ufunk.net.

Dream Messenger oil painting by Leszek Andrzej Kostuj with nanopore-like eyes. Taken from ufunk.net.

I don’t really know who first dreamt the exciting idea of moving DNA through a nanometer-sized pore to read sequence as electronic blip-like signals, but lots of folks are glad that someone did because this seemingly impossible dream is now reality.

My initial close encounter with this almost alien idea—pun intended—happened in 1998 when I first read about it in a patent by George Church (my recent blog-pick as The Most Interesting Scientist in the World), David Deamer, Daniel Branton, and others jointly assigned to Harvard and the University of California. This patent was filed 20 years ago on March 17, 1995 so St. Patrick’s Day should share with what I hereby suggest as annual Nanopore Sequencing Day.
Continue reading

Three Takeaways from the 3rd Next-Generation Sequencing Conference

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

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

Pacific Biosystems: direct sequencing of modified DNA

jonas

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

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

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

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

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

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

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

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

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

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

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

Nanopore sequencing:  small holes with big promise but bigger challenges

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

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

david

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

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

nanopore

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

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

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

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

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

eske

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

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

Just how old is ‘ancient’ DNA?  

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

horse

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

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

How old can you go?  

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

 

plot

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

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

As always, your comments are welcomed.