Nanopore-based Single-Molecule Spectroscopy

Nanopore-based Single-Molecule Spectroscopy

Michael Berger at the Nanowerk blog has an article on the latest efforts to use nanopores to characterize proteins and sequence single DNA molecules. His report focuses on successful experiments that demonstrated the feasibility of single-molecule DNA through-the-pore spectroscopy. On our pages, we wrote on a number of promising experiments in which scientists developed special nanopore channels, that interacted with translocating molecules and “reported” their molecular structure and sequence (see flashbacks below).

From the current Nanowerk article:

“In recent years, the creation of nanochannels or nanopores in thin membranes has attracted much interest due to the potential to isolate and sense single DNA molecules while they translocate through the highly confined channels” Dr. Joshua Edel, a lecturer in micro- and nanotechnology at the Imperial College London, explains to Nanowerk. “Nanopores for such applications have already been fabricated but in all studies to date, the detection of translocation events is performed electrically by measuring the ionic current” (what this means is that molecules translocating through a nanopore will momentarily perturb the ionic current, with the duration of the perturbation and the magnitude of the current blockade providing more detailed information about molecular shape and structure).

Edel’s group, together with collaborators from Drexel University, recently presented proof-of-concept studies that describe a novel approach for optically detecting DNA translocation events through an array of solid-state nanopores which allows for ultrahigh-throughput, parallel detection at the single-molecule level (“Single-Molecule Spectroscopy Using Nanoporous Membranes”).

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 ?-hemolysin (?HL), 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 ?HL 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 ?HL 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.]

Oxford Nanopore introduces DNA ‘strand sequencing’ on the high-throughput GridION platform and presents MinION, a sequencer the size of a USB memory stick

17th February 2012

- New generation of sequencing technology uses nanopores to deliver ultra long read length single molecule sequence data, at competitive accuracy, on scalable electronic GridION platform. Miniaturised version of technology, MinION, will make nanopore sequencing universally accessible -

17 February 2012, Oxford, UK/FL, US. Oxford Nanopore Technologies Ltd. today presented for the first time DNA sequence data using its novel nanopore ‘strand sequencing’ technique and proprietary high performance electronic devices GridION and MinION. These data were presented by Clive G Brown, Chief Technology Officer, who outlined the Company’s pathway to a commercial product with highly disruptive features including ultra long read lengths, high throughput on electronic systems and real-time sequencing results. Oxford Nanopore intends to commercialise GridION and MinION directly to customers within 2012.

Oxford Nanopore’s GridION system consists of scalable instruments (nodes) used with consumable cartridges that contain proprietary array chips for multi-nanopore sensing. Each GridION node and cartridge is initially designed to deliver tens of Gb of sequence data per 24 hour period, with the user choosing whether to run for minutes or days according to the experiment.

Oxford Nanopore will introduce a new model of versatile pricing schemes designed to deliver a price per base that is as competitive as other leading systems at launch. Further substantial pricing improvements are expected with future development to the technology, in particular with increases in nanopore processing speed and higher density electronic sensor chips.

Oxford Nanopore has also miniaturised these devices to develop the MinION; a disposable DNA sequencing device the size of a USB memory stick whose low cost, portability and ease of use are designed to make DNA sequencing universally accessible. A single MinION is expected to retail at less than $900.

“The exquisite science behind nanopore sensing has taken nearly two decades to reach this point; a truly disruptive single molecule analysis technique, designed alongside new electronics to be a universal sequencing system. GridION and MinION are poised to deliver a completely new range of benefits to researchers and clinicians,” said Dr Gordon Sanghera, CEO of Oxford Nanopore. “Oxford Nanopore is as much an electronics company as a biotechnology company, and the development of a high-throughput electronics platform has been essential for us to design and screen a large number of new candidate nanopores and enzymes. Our toolbox is customer-ready and we will continue to develop improved nanopore devices over many years, including ongoing work in solid state devices.”

Summary of presentation

At the Advances in Genome Biology and Technology conference (AGBT), FL, US, Oxford Nanopore presented:

A novel method of DNA ‘strand sequencing’ that uses an array of proprietary protein nanopores embedded in a robust polymer membrane. Each nanopore sequences multiple strands of DNA from solution in succession, as individual strands are passed through the nanopore by a proprietary processive enzyme. Base calling is performed by identifying characteristic electronic signals (disruptions in current through the nanopore), created by unique combinations of DNA bases as they pass through a specially engineered region inside the nanopore.DNA and enzyme are mixed in solution, engage with the nanopore for sequencing and once the strand has been completed a new strand is loaded into the nanopore for sequencing.

Genomes that have been sequenced as contiguous reads comprising both complementary strands of the entire genome. An example was shown of lamda, a 48kb genome, sequenced as complete fragments, whose sense and antisense strand total 100 kilobases. Read lengths mirror fragment sizes in the sample with no exponential loss of processivity.

Accuracy levels competitive with existing market-leading systems were shown. No deterioration of accuracy is seen throughout the sequencing of individual strands. A development pathway was presented that is expected to achieve accuracy exceeding current market-leading platforms through further design iteration of Oxford Nanopore’s custom-made nanopores.

Oxford Nanopore’s GridION platform was presented, consisting of a scalable network device – a node – designed for use with a consumable cartridge. Each cartridge is initially designed for real-time sequencing by 2,000 individual nanopores at any one time. Alternative configurations with more processing cores will become available in early 2013 containing over 8,000 nanopores.

Nodes may be clustered in a similar way to computing devices, allowing users to increase the number of nanopore experiments being conducted at any one time if a faster time-to-result is required. For example, a 20-node installation using an 8,000 nanopore configuration would be expected to deliver a complete human genome in 15 minutes.

A variety of sample preparation options were presented. No sample amplification is required and any user-derived sample preparation resulting in double stranded DNA (dsDNA) in solution is compatible with the system. With nanopores embedded in robust polymer membranes, dsDNA can be sensed directly from blood and in some cases with no sample preparation.

Oxford Nanopore’s disruptive “Run Until…” informatics workflow: Nanopores allow the analysis of data in real time, as the experiment happens. Each GridION node contains all the computing hardware and control software required for primary analysis of data as it is streamed from each nanopore, resulting in full length real-time delivery of complete reads so that the user can perform secondary analyses as the experiment progresses. This allows the user to pre-determine an experimental question and continue the sequencing experiment until sufficient data have been accumulated to answer the question and move on to the next experiment.

Oxford Nanopore intends to introduce a new pricing model for its GridION sequencing system, which moves away from the traditional instrument price and consumable price. This is designed as a series of packages that allow the user to tailor a scheme to their budget structure, whether more flexible with capital or consumable expenditure. Transparent pricing schemes are designed for online ordering and fulfilment, with discounts applying to larger packages. Overall the schemes are designed to deliver a competitive ‘price per base’ compared to other systems on the market based on like-for-like user settings.

Further information is available at the Company’s website www.nanoporetech.com. While orders are not yet being taken for the GridION and MinION systems, interested users may register their interest at the website.

-ends-

Contact: Zoe McDougall, Communications. media@nanoporetech.com

Notes to editors

Oxford Nanopore Technologies Ltd is developing a novel technology for direct, electronic detection and analysis of single molecules using nanopores. The modular, scalable GridION technology platform is designed to offer substantial benefits in a variety of applications. The miniaturised MinION device is the size of a USB memory stick, designed for portable analysis of single molecules. Oxford Nanopore intends to commercialise GridION and MinION directly to customers for DNA ‘strand sequencing’ in 2012.

In addition to DNA sequencing, the system is also compatible with the direct analysis of RNA. Oxford Nanopore is also developing a Protein Analysis technology that combines target proteins with ligands for direct, electronic analysis using protein nanopores. These nanopore sensing techniques are combined with the Company’s proprietary array chip within the GridION system and MinION.

The Company is also developing the subsequent generation of nanopore sensing devices based on solid-state nanopores.

Oxford Nanopore has licensed or owns more than 300 patents and patent applications that relate to many aspects of nanopore sensing including fundamental nanopore sensing patents, analysis using protein nanopores or solid state nanopores and for the analysis of DNA, proteins and other molecules, including the analysis of probe molecules on DNA. The Company has collaborations and exclusive licensing deals with leading institutions including the University of Oxford, Harvard and UCSC. Oxford Nanopore has funding programmes in these laboratories to support the science of nanopore sensing. This includes the use of functionalised solid-state nanopores for molecular characterisation, methods of fabricating solid-state nanopores and modifications of solid-state nanopores to adjust sensitivity or other parameters.

Source : http://www.nanoporetech.com/news/press-releases/view/39

Related Posts Plugin for WordPress, Blogger...
Be Sociable, Share!

About the Author

has written 1822 posts on this blog.

Copyright © 2017 Medical Technology & Gadgets Blog MedicalBuy.net. All rights reserved.
Proudly powered by WordPress. Developed by Deluxe Themes