More Blogging from #ASHG14: The San Diego scene

It has been almost a week since ASHG14, but I forgot to link the last blog I wrote for SDBN on the meeting. This was all about the smaller and emerging genomics-based companies in the San Diego area. The write-up covered the biotechs that had presence in the form of booths at the meeting. Hence the lack of mention for entities like AllSeq.com and the stealth-mode Omniomics.

In many ways, San Diego is the perfect location for the ASHG 2014 annual meeting, and not just for the perfect weather and the beautiful convention center. The area is home to the two top genomic technology leaders, Illumina, and Life Technologies (now Thermo Fisher). Apart from these powerhouses, a large number of smaller biotechs in the area also deal with genomics. Yesterday, I listed several of them represented at the meeting as exhibitors: Cypher Genomics, which provides specialized bioinformatics services, Edico Genome, which recently launched the first microprocessor dedicated for genome sequencing, Genection, which is developing a NGS panel for testing mutations in AML, and Pathway Genomics, which provides a host of genetic tests related to personalized health. I forgot to add to the list, BioNano Genomics, which has a platform for detecting structural variations in human genomes. With such a variety of exciting technologies, it seems appropriate to devote a blog post focusing on these upcoming genomic businesses.

 

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Blogging from #ASHG14: Day 2

Day 2 of the ASGH14 meeting at the SDBN blog.

Link to post here.  Excerpt:

The second day of the ASHG 2014 meeting once again featured a number of very exciting concurrent sessions. I was torn between attending the rather cute, and appropriately named ‘Cloudy with a Chance of Big Data’, ‘Population Structure, Admixture, and Human History’, or ‘Impact of Human Knockout Alleles’. I had already covered a bit about Big Data, so the last two seemed more interested. In the end, the human knockout alleles won out, not in small parts due to the fact that the first speaker was Daniel MacArthur of Harvard University, who is among the very prolific scientists on social media (@dgmacarthur).

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Blogging from #ASHG14

The American Society in Human Genetics 2014 annual meeting is in town, and I am covering it as a blogger for San Diego Biotechnology Network.

Here is the first post with focus on big data and crowdsourcing genetics.

The field of human genetics has been revolutionized in the last few years by the falling cost of genome sequencing. The first human genome sequencing was a $3 billion effort involving 200 scientists over 11 years. The latest sequencing machine launched by Illumina earlier this year, HiSeq TenX, can sequence human genomes for as low as $1000 within a week. One of the consequence of inexpensive sequencing has been the tsunami of data that need to be analyzed into clinically relevant information and outcomes.

Therefore it was quite appropriate that one of the first session in the morning was focused on the big data problem. The Distinguished Speaker’s Symposium, titled ‘Separating Signal from Noise’, consisted of Ajay Royyuru of IBM, David Glazer of Google, and Muin Khoury of CDC, reflecting the cross-disciplinary effort required to tackle these problems.

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AGBT Ahoy 2013 Edition

It is that time of the year again, when the blog tends to come alive with the annual AGBT conference at Marco Island, FL.  This is my third consecutive year, and hopefully I will keep alive the trend of significant blogging and tweeting from the conference (besides writing a meeting report for a journal). There is a sense of deja vu as well, as I was writing a similar post last year waiting for a red-eye flight to get me in time for some of the Wednesday talks (but thankfully, this year’s AGBT registration was  much more hassle free – no gone in sixty second excitement).

There has been some murmurs about the agenda being a bit less exciting as well. I guess the PacBio, Illumina, Ion Torren, Oxford announcements  of the last several years have really spoiled us. I have to agree that the talks are a little short on ground breaking genomics technology (although there are a few single-cell genomics techniques that I am really interested in). However, the trend of greater focus on clinical genomics that I noticed last year continues. In fact, day 1 is fully devoted to clinical applications. As Erica Check Hayden writes, this is reflective of a general trend in the genomics world.

The only other sort-of-excitement is with NABSys, who are demonstrating their ‘positional sequencing’ technology based on solid-state nanopores. Keith Robison got a preview and wrote about it on his blog.

Irrespective of the agenda, I am still quite excited for the meeting. Also hoping to run across some of the people I have seen and ineracted with on Twitter during the many networking. opportunities and the (in)famous parties.

 

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Elaine Mardis on Next-Generation Sequencing

If you are interested in learning more about ‘next-generation DNA sequencing’ technologies (i.e all post-Sanger sequencing methods),and its applications do not miss this lecture by Elaine Mardis of the Genome Institute at Washington University. It is a long talk, but is exhaustive and delivers all the important details on this topic.

The talk was part of NHGRI’s Current Topics in Genome Analysis 2012 series. Check out the full schedule here. It is great that the lectures are video-taped and uploaded on Youtube.

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I see your 8K and raise you a million….

Few days ago when Kevin Davies headlined his article about Oxford Nanopore Technology’s announcement at AGBT with words like ‘ nanopore wars’, I thought it was bit of a hyperbole.

Apparently it is not. Less than a week after ONT’s CTO Clive Brown’s widely hailed talk, the CEO of Genia Technologies, one of the latest entrant to the nanopore sequencing arena, said the company will come up with a sequencing device by next year that will contain ‘up to 1 million nanopores on a single integrated circuit’! He was speaking at the Molecular Medicine Triconference underway at San Francisco.

(No word on whether he lifted his left little finger close to his mouth while he announced the ‘one million nanopores’)

The key difference between Genia and ONT is that the latter is mechanically placing the pores on the chip, while the former is using a CMOS chip.

Linking each pore to the chip in a mechanical manner doesn’t really scale, Roever said. “If you put [pores] on an integrated circuit, the electronics are embedded in the chip under the electrode… The only way to get to this density is if you put electronics on the chip.”

So Genia’s approach involves using semiconductor chips with electrodes embedded in each well where protein nanopores can be inserted on lipid membranes.

Apparently they can form lipid bilayers (they use natural lipids unlike the synthetic polymers from ONT) and also insert the protein nanopore in an automated manner. No word on the nature of the protein nanopore they are using. While I seem to recall from an earlier story that their platform was supposedly ‘chemistry independent’, it is likely they are using α-hemolysin as well.

The immediate implication of the denser packing of nanopores is  increased throughput and hence higher coverage of genomes in lesser time. However, they are planning to insert the protein from the solution, which means they are going to be up against Poisson limitations of how many wells will possess a protein. I believe the limit is 38%, which means they will still significantly have more pores than the 8K chip from ONT.

If there was skepticism about ONT, who demonstrated real sequencing data, albeit on a short viral sequence, you can double that for Genia. The only data they seem to have shown is the ability to pull through 50,000 strands of DNA.

Still, Genia is well funded, with Life Technologies having a strategic investment on the company. So they might very well deliver the goods as promised.

Meanwhile, this so-called ‘war’ between ONT, and Genia and other nanopore technologies should also provide errr….. holesome entertainment in the next couple of years.

As such, along with GnuBio and LaserGen also announcing commercial release of their products later this year, these are exciting times for sequencing technology.

 

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Beyond the Hype – The Oxford Nano Announcement at AGBT

As predicted, Oxford Nanopore Technology (ONT) made the biggest splash at the recently concluded Advances in Genome Biology & Technology meeting. There may have been apprehensions that Oxford wouldn’t live up to the pre-conference hype, but a full house of meeting attendees – plus loads of people following on Twitter – were not disappointed.  Following the Steve Jobs school of product launch (website down on the morning of the talk and then a new one up just after the announcement, Clive Brown making a dramatic pause before announcing ‘one more thing……’) ONT presented a novel nanopore sequencing technology that caused audible gasps in the audience, hijacked all other discussions for the rest of the meeting, and was immediately being hailed as a ‘game-changer’.

Enthusiastic approvals also streamed through the Twitter feeds, and equally glowing reviews came in the form of blog-posts [1] almost immediately (by those who had been given early access to the presentation). The paradigm-shifting promise of an USB-powered, almost palm sized device that can sequence billion bases of DNA on your laptop has captured everyone’s imagination, with main-stream media like New York Times covering the story. The announcement rapidly affected the stock prices of other players in the sequencing field as well (suited financial types attending the meeting were observed to be furiously texting as the talk was progressing).

Many blogs and news-sites (check the list [1] below) have extensively covered the various features of the two sequencing platforms announced by ONT – the GRIDIon and the MINIons, as well as how it might affect the sequencing landscape (though unfortunately in some cases, the posts sound like rehashing of marketing materials). So I won’t go into those details here.

However, I would like to go a little beyond the hype, briefly recapping the talk by Clive Brown to analyze some of the scientific breakthroughs made by ONT that enabled the technology at the heart of these sequencing devices.

[As an aside, my pre-announcement predictions of the technology was off on many of the details, though the general ideas were as expected. Of course, the MINIon totally came from the left field.]

Basically the platform involves a chip containing densely arrayed nanopores, the electrical current through each of which can be read separately. DNA can be added to the chips with an enzyme and sequencing is performed as the enzyme bound DNA is pulled to the pore.  The only requirement for sequencing to work is that the DNA should have a 5’- overhang. However, there are number of other DNA forms that will work as well. The two sequencing runs demonstrated during the talk were performed with DNA where a hairpin is added at one end (possibly so that the DNA is arrested at the top of the pore and can be run through the pore in the reverse direction once more).

Zooming into the sensor itself, the protein nanopore being utilized by ONT is still α-hemolysin (αHL), but it is an engineered protein with several amino acids within the pore mutated to other residues to improve base-discriminating abilities.

Rather than using a polymerase to control the speed of the DNA (as I was thinking), they have developed a novel motor-enzyme for the ratcheting motion. However, they would not say which particular protein acts as the rachet other than that it is certainly not a polymerase. Not having a polymerase is actually useful since additional nucleotides do not need to be added to the solution and the DNA is available in the original form for re-sequencing if needed.

For obtaining both the optimal pore and the motor-enzyme, the ONT scientists had to screen hundreds of mutations to hit upon the perfect ones.

ONT has also done away with the traditional lipid membrane that the αHL protein is usually embedded in, and replaced it with a robust synthetic polymer. The protein-polymer combination is preloaded on the chips and is extremely stable with 80% of the pores still functional after three days. It also has the ability to withstand dirty samples like blood and sewage wate. This I believe is actually a major material science-biochemistry interface innovation. While αHL is a relatively stable protein on its own, if the synthetic polymer can be adapted to other proteins, it could be useful to protein arrays in general. But it is mainly the stability of this polymer membranes (and to some extent the electronics) that enables the disposable USB drive-sized MINIon sequencer.

The synthetic polyemer-protein interface is combined with their own low-noise integrated circuit to produce the dense array of protein pores, each an individual sequencing machine, on their chips.

For actual base-reads, ONT still has not achieved single-base sensitivity (though Brown did mention they are working on it). Instead they are reading three bases at a time, leading to 64 different current levels. They then apply the Viterbi algorithm – a probabilistic tool that can determine hidden states – to these levels to make base calls at each position.

Using this technology, ONT was able to sequence two smaller sized genomes – a phiX viral DNA (5kbase) and the lamda DNA (48kbase).  In both cases, DNA was sequenced as a single linearized fragment. Each fragment was read twice, once in each direction. The error was found to be ~4%, and mainly caused due to the nature of the predictive nature of base-calling, and fluctuations in currents due to DNA vibrating in the pore. The scientists at ONT are working on further pore mutations to remove this noise.

Considering that many different groups have struggled for over 25 years to produce sequencing information using nanopores, the presentation of this data is without doubt a significant scientific landmark in this field. The scientific team at ONT deserve rich kudos for making it happen. It was also heartening to see David Deamer and Dan Branton, two people from the group that were the first to envision nanopore sequencing, take in the talk from the front row. They must have been incredibly excited.

[One should mention here that Jens Gundlach of University of Washington, Seattle, also had a poster at the meeting that showcased nanopore sequencing data – albeit on a much shorter, 20-30 basepairs, scale – using the MspA protein and a phi29 polymerase enzyme].

In summary, Oxford Nanopore seems to have solved the three key technical challenges faced by protein nanopore sequencing technology: controlling speed of DNA, fragility of the biological membranes, and the lack of sharp sensing zones. They have demonstrated proof-of-principle of their pore by sequencing the phiX and lambda DNA at a relatively (compared to say, PacBio) high accuracy.  They have unveiled a conceptual design of devices that will contain a dense array of protein pores that will work in parallel to sequence DNA, which they say will be available to customers at very competitive pricing (for GRIDIon) or unprecedented portability (MINIon). Combined the potential of unprecedented long reads, possibly upto 1000kB, no expensive or time-consuming library preparation, and potential direct RNA reads and epigenetic detections, the technology is indeed a massive game changer.

Well……..theoretically.

Until ONT demonstrates actual sequencing of a more complicated genome (a microbial one at minimum), there will be a healthy degree of skepticism. The reaction from Jonathan Rothberg, inventor of the 454 and Ion Torrent sequencing technologies, comparing the MINIon to ‘cold fusion’ might be a bit extreme. However, a majority of scientists I spoke to at AGBT agreed that while ONT’s technology is very promising, the proof will come from real world usage of the devices. The exceptional promises made by Pacific Biosciences two years ago, which they have only partially delivered on, is on everyone’s mind.

According to this post, ONT is providing about a dozen institutes with machines for beta testing, so hopefully we will have some real data very soon. Additionally, given the $1000 price range, I expect quite a few labs all over the world will buy a MINIon for simply testing the technology.

Still, the lack of data or some scientific details at the talk is a bit bothersome. I did not exactly see data that demonstrates that 64 levels of currents are being detected. Additionally, I was not quite sure if the phiX or the lambda DNA was sequenced using just one pore or the actual sensor array. There was mention of rabbit blood and waste-water being added to the chip for sequencing. This is very impressive in that the protein-polymer was not affected, but did they obtain actual sequencing data from these experiments? I do not recollect seeing that. Finally, while the sequencing of phiX or lambda DNA was quite exceptional as mentioned, one will have to wait for real sequencing data on complicated genomes to find out base length reads, error rates etc.

To be fair, it was a very short talk (20 minutes). But perhaps they could have released more data at the poster. Or allowed people to download some early data from their website.

In addition to these concerns, it seems to me there are couple of issues with the sequencing methods as it stands.

Firstly, since DNA is not amplified or modified, there will be modified nucleotides that will have a different current level. For future sequencing with real genomes, this will undoubtedly add to the complexity of base calling since the number of current levels being detected will be much higher than 64.

Secondly, if the DNA is added without any preparation, varying DNA lengths could cause some issues. Shorter DNAs are more likely to be pulled to the pore due to faster diffusion leading to a bias in the sequencing. I expect there will have to be some sort of fragment sizing, and there are quite a few commercial instruments out there (e.g the Pippin technology from Sage that was on demo at the conference) that can do this.

As such, the next few months will be extremely interesting as more details and data emerge confirming if ONT have indeed found one of the Holy Grails of sequencing (and yes, Brown showed an image of the rabbit from the Python movie, very appropriate).

[The ONT announcement completely overshadowed some other interesting technology news at the meeting, including Ion Torrent’s Ion Proton machine, some new data on very long reads from Illumina on their machines, and two other prospective next-generation sequencing technologies from GnuBio and LAserGen. More unfortunately, it overshadowed some really interesting basic scientific talks presented there. I will try to get a brief review of those very soon.]

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[1] Further reading:

a. Kevin Davies at BioIT World possibly has the most comprehensive description of both the individual sequencers and the complete systems being offered by ONT. A must read, since he offers much more details than I have on how the GRIDIons can be stacked to produce sequencing information at a faster pace, as well as details on the costs etc.

b. Both Ketih Robison of Omics! Omics! and Nick Loman of Pathogenomics got to speak to the ONT team the night before the announcement and had posts up pretty quickly after the announcement.

c. Erica Check Hayden at NatureNewsDuncan Graham-Rowe at NewScientist , and Luke Jostins at Genomes Unzipped have covered the story very well as well.

Also read Ellen Clark’s summary.

One more general story from Luke Timmerman of Xconomy

d. The financial publications covered the announcement as well. Here is a Bloomberg story, and Matthew Harper broke some of the early reactions from the market, as well as Rotheberg’s statements.

(do let me know if there were any other blogs/news articles about the Oxford Nanopore technology, and I will add a link here).

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AGBT ahoy!

So here I am at the San Diego airport waiting for my red-eye flight which will get me into Marco Island, FL tomorrow morning – via Charlotte, NC, Fort Meyers, FL and a hour long bus drive – for the AGBT meeting. Still feels a bit unreal since two weeks ago, I wasn’t even going to attend (hence the last minute red-eye, something I try to avoid always) .

Last year’s meeting (my first) was enjoyable in many respects and I expect no less this time around. Particularly exciting is the anticipation for Oxford Nanopore’s talk on protein-nanopore sequencing - as I wrote about yesterday. This morning, I also read that NABSys will be showing (re)sequencing data from their solid-state nanopore device. Therefore, much to look forward to at AGBT from the point of view of nanopore sequencing.

Meanwhile, we will also get the first glimpse of the new Ion Torrent device, the Ion Proton Sequencer. Life Technologies has been heavily promoting their Ion bus – where the science and partying is supposed to go together (somewhat of a theme at AGBT going by last year).

There should also be a few talks and posters on the application of third generation sequencing devices like the PacBio SMRT technology, Ion Torrent’s PGM, and Illumina’s PGM that were introduced in the last two years and have had a chance to be tested in the real world.  Plus there will be plenty of other exciting genomic science for consumption. Kevin Davies has a very good preview of what to look forward to in Florida (as well as a brief history of this meeting), and there is another preview at the MassGenomics blog.

I will try to tweet (via @omespeak) breaking news as much as possible from the meeting. Thankfully, AGBT has adopted a very progressive (and clear-cut) social media policy this time around, so expect much action at the #AGBT hashtag.

 

And do say hi if you are there at Marco Island as well!

 

 

 

 

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On Oxford Nanopore’s ‘disruptive’ technology

(Apologies for the bad formatting with the punctuations, seems to be an issue with this theme. I am trying to fix it)

The annual Advances in Genome Biology and Technology (AGBT) meeting commences at Marco Island, Florida in a few days. Quite easily one of the most popular venues for genomics (the 2012 meeting was sold out in the first two minutes of opening of registration), the conference has gained a reputation for showcasing cutting-edge DNA sequencing technologies. Past years have witnessed the introduction of major sequencing innovations like Ion Torrent’s Personal Genome Machine, and Pacific Bioscience’s single-molecule SMRT sequencing at this meeting.

This year, the major pre-conference buzz has been over a presentation scheduled for Friday Feb 15th from the CTO of Oxford Nanopore Technologies (ONT) titled “Single Molecule ‘Strand’ Sequencing Using Protein Nanopores and Scalable Electronic Devices”. A corresponding press release from the company talks of revealing ‘disruptive features’ at the meeting and commercial release of ‘DNA strand sequencing products’ later this year.

ONT has been one of the several companies competing in the so-called fourth (or ‘next-next’) generation sequencing technology field. Such technologies are expected to lead to faster and cheaper sequencing of human genomes – in a few hours and for few hundred dollars rather than the weeks and few thousand dollars that current technology supports. Cheap sequencing, it is hoped, will eventually lead to more widespread use of genomics for human health.

Given the promise of nanopore sequencing, and the relatively teasing nature of ONT’s press release, there has been much speculation on the interwebs about the nature of their device, its costs, performance parameters etc. The most comprehensive discussion on this topic is at Dr. Keith Robison’s blog (highly recommended read). While I agree with much of his conjectures, his post is looking at the device mainly from the point of view of genomics applications. Over here, I would like to cover some of the more basic science, and methodology aspects, outlining the challenges of nanopore sequencing and how ONT might be tackling them.

Background & Key Bottlenecks:

But first, a brief background on nanopore sequencing. This single-molecule sequencing approach involves threading single-stranded DNA (or RNA) molecules through a nanometer sized pore (that could be biological or solid-state) under a voltage bias. The ionic current through the pore generated by the electrical bias is blocked by the moving DNA, with  (ideally) each base in the DNA producing a different level of block. Thus measurement of these blocking levels can produce the desired sequence information.

The attraction of such a simple platform is easy to comprehend. Both reagent and instrument costs are much lower compared to current methods that require modified bases, expensive cameras etc. It is single-molecule method that does not require amplification to clusters of DNA for sequencing. This leads to reduced error, shorter overall sequencing time and is useful for directly detecting epigenetic modifications. Finally, read lengths can be quite long, potentially in the kilobase range, thereby reducing bio-informatics burden.

While the method has been around for a long while (the idea of protein nanopores being used for sequencing was floated around the mid-90s, much before the human genome was even sequenced), thus far, no one has truly demonstrated actual sequencing of a moving DNA strand through a pore. Two major technical challenges have prevented this method from taking off: (a) Under electrical biases used to pull the DNA through the pore, the motion is too fast to adequately resolve each base with accuracy. (b) Lack of a sharp sensing zone in the pore, such that as the DNA is threading through, only a single base produces the majority of the blocking current at a time. Multiple bases adjacent to each other producing a combined blocking current will lower the resolution.

The published result that comes closest to demonstrating nanopore sequencing is from Jens Gundlach’s group where they used a protein nanopore, MspA. However, it was not a direct sequencing of a DNA strand; they had to amplify the DNA to convert each base into a corresponding three base (usually homopolymer)s sequence and insert a 14-mer double-stranded sequence between the homopolymers e.g. ATC was converted to X14AAAX14TTTX14CCC etc. The double-stranded section being too wide to enter the pore, halts the progress of the DNA (thus taking care of the speed issue) while the three base ‘code’ is read at the sensing zone (overcoming the issue of a wide sensing zone). To read the next base, a higher bias was applied to unzip the double-stranded section, resulting in the next three base unit occupying the sensing zone. As such, it was a proof of principle study demonstrating the sequencing of a short 5-base sequence. But the requirements for amplifying DNA reduces some of the simplicity of nanopore sequencing.

The ONT ‘strand’ [1] sequencer:

Details about the technology are scarce from ONT, but one can make educated guesses based on recently published works and talks from Hagan Bayley’s group at Oxford University, the lab from which ONT was spun-off (and apparently is the first technological spin-off from that hallowed university), as well as their collaborators in various US universities.

The Pore: The pores for nanopore sequencing can be either biological (a protein ion channel) or solid state (holes punched on silicon surfaces, and more recently, on graphene). The advantage of the protein pores over solid-state ones is that they possess reproducible dimensions and can easily be modified genetically or chemically to enhance interactions with the DNA.  The disadvantages are that protein pores require a lipid bilayer environment which have the potential to break down easily.  While ONT is said to be working on solid-state pores for the future, their immediate product will certainly involve biological pores.

Oxford’s go-to protein over the years has been α-hemolysin, which is naturally secreted by the pathogenic bacteria Staphylococcus aureus, but also has been produced in the lab and engineered extensively. Majority of the tinkering with the protein has been performed in the laboratory of Dr Bayley. However, I would not rule out their using of the other protein nanopore, MspA, described above. At the last AGBT meeting, it was mentioned that Dr Gundlach was collaborating with Dr Mark Akeson who is on the advisory board of ONT and a close collaborator.

Controlling DNA speed: It was also at last year’s AGBT where Dr. Akeson presented results on overcoming one of the key challenges of nanopore-sequencing: slowing the speed of DNA motion. Dr. Akeson and colleagues successfully demonstrated control of DNA motion by ratcheting it through the pore using a special polymerase. The technique involves pulling a DNA-polymerase complex into the pore; since the polymerase is larger than the size of the pore, it gets blocked at the entrance. Subsequently, as the polymerase adds a nucleotide the single DNA strand gets pulled through the pore for sensing. While no sequencing data was presented last year, the passage of a sequence of three consecutive abasic residues through the α-hemolysin pore was demonstrated. Additionally, In response to a question, Dr Akeson had mentioned that actual sequencing data was just ‘a few months away’.

Therefore, it is very likely that ONT’s nanopore platform will involve the use of such polymerases to thread DNA through either α-hemolysin, or MspA. Do expect the polymerase to be engineered such that it has a longer lifetime, and can function under the higher salt concentrations usually used during nanopore sequencing (higher salt means higher ionic strength and hence higher current levels which aid is base resolution).

Sensing zone: This is a major issue and is a little trickier to figure out how ONT may have solved it. The MspA protein has a natural advantage compared to α-hemolysin due to its shorter pore length resulting in a relatively sharper sensing zone. Still, the best resolution currently seems to be at three-peats or more of nucleotides, based on published data. Enhancing pore detection has been attempted by either mutations, or by addition of chemical moieties. It is possible that ONT may have made a major discovery here (and it is a major scientific breakthrough if they have) that produces single-nucleotide sensing zones in either protein. If this is the case, it is also likely that any such modifications to the pore will be kept confidential for now.

In absence of such breakthroughs, it is also possible that combinations of bases (e.g a three base sequence) can be read instead of a single base, but would require the ability to discriminate more than four blocking levels. The Bailey group have also previously published on the possibility of using two sensing zones in α-hemolysin to improve base detection.

An alternative approach could involve amplifying the DNA (in manner similar to that by Dr. Gundlach, and also employed by Dr. Amit Meller and colleagues for their optipore sequencing approach) such that each base corresponds to a homopolymer sequence of three or more bases that can be resolved more readily.  Such amplification steps will however add considerable time (and cost) to sequencing and could also induce errors during the preparation steps.

Multiplexing: As such, read errors will happen during nanopore sequencing due to slipping of the DNA, polymerase sometimes moving the DNA too fast, inability to read homopolymer stretches in the sequence etc. Additionally, if the DNA is slowed down by polymerases, total sequencing time will be lengthened for each pore.  To overcome these issues, the sequencing will have to be multiplexed. This will most likely be achieved by the modular GridION system, announced by the company about a year ago. Each GridION module can accept a chip that contains multiple wells for nanopores. Several modules can be clustered together as well for higher-throughput. However, it is unclear how they can control pore formation at each well or prevent electrical cross-talk between the wells.

Bioinformatics: I don’t have enough expertise on this topic to comment on what sort of informatics support will be required. If the base resolution is low then bioinformatics of course becomes critical to piece sequence information from the multiplexed sequencing processes.

Other Issues: Other challenges include making sure the lipid bilayers, which contain the protein pores and are fragile (they can break due to mechanical vibrations), are held stable through the duration of the sequencing. The protein also sometimes leaves leaves the bilayer or one could have multiple insertions of the protein on a single bilayer. Additionally, DNA is double-stranded, and these nanopores perform single-strand sequencing. So there is the challenge of keeping the DNA single-stranded – this could either be through the use of high pH (which will certainly affect the chemistry of the probe) or by separating the strands in a preparation step. However, these problems – though not insignificant – are more of ‘engineering’ issues that can be overcome by employing various tricks rather than a basic scientific barrier.

Disruptive Potential:

Overall, the disruptive potential of ONT’s technology will depend on how many pieces of the nanopore puzzles, as outlined above,they have managed to solved. If they have indeed made key discoveries that allow unamplified DNA to be sequenced with long-read lengths at low error rates, it will be highly disruptive. This will mean, as Dr Robison points out, a system similar to Pacific Biosciences but possibly with low error rates, and quite certainly, much cheaper. But if it requires DNA amplification steps or has high error rates, the application will remain niche in the short term.

I would not like a hazard any guesses either way. As such, we will find out more in a few days and I am extremely excited to be able to head to AGBT to get these answers live (I am also quite lucky since I missed the first registration attempt and found out only a week or so ago that a spot had become available!).

Announcements from ONT, as well as other interesting science at the meeting, will be updated both on this blog, as well from my Twitter account @omespeak. Do follow if interested.

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Note:

1. One should make clear that the ‘strand sequencing’ approach that is supposed to be discussed at the AGBT meeting is distinct from Oxford’s other nanopore approach, which involves chopping the DNA into individual nucleotides that can be uniquely detected in the pore. ONT has a signed agreement with Illumina for marketing that particular technology. Proof-of-principle of this method was published in 2009, but it does not sound like the technology is ready yet. The major issue there is to make sure each base, as it is chopped the enzyme definitely enters the pore, and not diffuse away – a nontrivial task.

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Disclosure: I work for a company that is developing protein-nanopore based sequencing applications as well. However, the views expressed here are solely my own and does not represent any opinions or policies of my employer.

 

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Linked In: Challenging the Central Dogma

A study led by researchers at University of Pennsylvania in Philadelphia, published last week in the online edition of Science, has found a large degree of mismatch between the RNA and DNA of 27 individuals. While it is known that RNA transcribed from DNA often gets edited by post-transcriptional processes (leading to a different protein sequence than what was coded by the DNA), the scale and nature of editing found here (tens of thousands of changes) is unprecedented, and also suggest some yet unknown editing  machinery.

As expected with results that directly challenge well-established paradigms in science, there has been a healthy dose of skepticism and caution amongst scientists. As  Erika Check Hayden reports in NatureNews:

[M]ost scientists contacted by Nature remained cautious about the significance of the finding and its possible impact on biology. Some say it is possible that technical errors could have caused the results. For instance, high-throughput sequencing machines can make systematic errors in DNA and RNA sequencing experiments.

With respect to the systematic errors caused by sequencing machines,  Joe Pickrell has a lengthy post on Genome Unzipped on possible sources of such errors.

First, mismapping of reads in paralogous regions could lead to false signals of RNA editing. These false signals would even be replicated in follow-up experiements like those done by Li et al. (2011), because the two forms of RNA and protein are indeed present in the cell. However, the two forms of RNA and protein do not come from the same DNA sequence, and thus are not evidence of RNA editing. Second, mapping biases around splice sites (and other sorts of insertions/deletions in the genome) will cause mismapping and false inference of RNA editing.

Erika Check Hayden also has a follow-up story on NatureNews today delving into further details about the debate.

A more comprehensive look at the paper will have wait for another time (plus I have little knowledge in this area), but Ed Yong as usual has a very good post explaining the study (he has also helpfully collated reactions to the paper on Twitter from various scientists and journalists).

As such, if the conclusions from the study hold, this is an exciting result that seems to add another level of complexity to how our genomic blueprint translates into actual function.

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