MinION is almost here...

Breaking news: AGBT Feb 14th: David Jaffe from the Broad Institute is presenting a talk titled: “Assembly of Bacterial Genomes Using Long Nanopore Reads”. My assumption is that this is ONT, it may not be but see you there anyway.

Will anyone get his or her genome sequenced at AGBT this year?

Given HiSeq 2500 and Ion Proton it is entirely possible for someone to sequence their genome in the Illumina or Life-Tech booth’s at AGBT, so will anyone attempt it?

Nanopore sir? should be delivered in 2014 or perhaps 2030!

Many of the readers of this blog will have seen the announcements from Oxford Nanopore Technologies on their MinIon early access program. The people I talk to are almost universally excited; if a little sceptical about how quickly we’ll be getting rid of our HiSeq’s and Proton’s.

MinION early access program

Update from GenomeWeb at the bottom!

Get ready for millions of minions!  ONT have announced an early-access program for the MinIon (GridION later) and it is almost free to access. I'm sure they will see huge demand from eager NGS users. But who will have projects best suited to the MinION technology and who will be first to publish?

Let's not forget ONT's technology promises long-reads, how long is not completely clear but some applications will benefit more than others.

$1,000 buys you a MinION system, free flowcells (to an undisclosed limit), free sample prep and free sequencing reagents. Of course there is no such thing as a free lunch and ONT will require users to sign their End User License Agreement "to allow Oxford Nanopore to further develop the utility of the products, applications and customer support while also maximising scientific benefits for MAP participants". And the press release does give a lot of hope that ONT don't want to restrict your right to publish.

"MAP participants will be the first to publish data from their own samples. Oxford Nanopore does not intend to restrict use or dissemination of the biological results obtained by participants using MinIONs to analyse their own samples. Oxford Nanopore is interested in the quality and performance of the MiniION system itself."

I've signed up already!

You can too at by visiting the ONT contact page and selecting the box marked 'Keep me informed on the MinION Access programme'.

Update: Some more details came from GenomeWeb a few minutes after I posted. According to their coverage read-lengths may be up to 100kb but the number of pores could be as low as 500. This is exactly the kind of detail we are going to need to determine the best applications to run tests on.

Clive Brown and MinIon spotted at the cinema

So the drama is finally over and now we can expect great things from the company developing the worlds best nanopore sequencer, Illumina's Jat Flatley confirms they have one in development!

And at the same time MinIon is about to make it's comeback in the latest sequel from Universal...

Finally a nanopore sequencer that works

Today I was given an exclusive preview from the newest nanopore sequencing company on the planet, Norfolk Nanopore Technology. The new "Polonopore"technology has been incubating in the Norwich Research Park BioIncubator under the same roof as TGAC.

NNT's brand spanking new nanopore sequencer could mean the end for the HiSeq, MiSeq, Proton and PGM platforms and paves the way for $100 personal genomes. NNT are suggesting very high yields from long-reads using Polonopore technology. $100 for 100x coverage of 100 genomes in 100 hours.

Oxford Nanopore chip announced!

Almost...

I had a very enjoyable trip to the Science Museum in London this weekend and whilst there was amazed to see an Oxford Nanopore DNA sequencing chip on display.

The word on the street after ONT's AGBT 2012 extravaganza has been more like a quiet grumble about progress, the hype has certainly not been lived up to, now that quiet grumble is beginning to get noisier! Everyone was hoping for their MinION USB sequencer and the Star-Trekesque possibility of truly personal genome sequencing. Although even Jim McCoy didn't have that on his tricorder.

There is not even the tiniest hint of what is being done with academic collaborators, no posters, no papers, no seminars. Now all we can do is ponder on why the technology has not been launched as planned. Surely it was not pure hype to whip up interest in their last round of funding? Perhaps the technology has suffered issues, there is a lot going into what ONT and others are trying to do; hardware, software, enzymes and just about everything else all need to be designed and tested and any one of these could stop the system from working. Legal wrangling is almost certainly an issue with a huge number of patents in this field and companies like Illumina working on their own Nanopore technology.

So here it is: The blurb reads "Oxford Nanopore chip. Tiny pores 10,000 times smaller than a human hair sit in microscopic holes covering the surface of this speedy chip. DNA is read electronically as it zips through each pore, generating DNA sequence data."

Photo of ONT Nanopore chip in the "Who Am I" gallery at the Science Museum, London.

Sorry the picture and text are so crummy.

I'm sure we'll see the real thing soon, hopefully long before the 23rd century!

PS: The ONT chip is next to an ABI SOLiD instrument. Don't read to much into it being next to a technology that is already obsolete !

Oxford Nanopore did not disappoint

AGBT is over as far as many attendees are concerned, the sequencing landscape looks like it might be about to ramp up to the next gear. Again!

The new MinION (read about the USB seqeuncer later on) will cost $500. assuming everyone at AGBT buys one ONT get $5M in rvenue form one conference. This is before anyone does anything real with the technology.

At least one lucky punter got a sneak preview. Nick Loman, my collaborator on the Google Map of seqeunceres, got an early interview with Clive, Zoe and Dan at ONT. Read his post here.

Clive Brown from ONT presented “Single Molecule ‘Strand’ Sequencing Using Protein Nanopores and Scalable Electronic Devices” at AGBT in a 20 minute slot. It was 20 minutes with lots of information, much of it jaw dropping if it delivers as well as everyone hopes. The company also have a press release which says “[Clive] 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".

I can't resist adding this image of Clive... I lifted it from Bloomberg (sorry Bloomberg). He looks like the cat that killed the rabbit! And yes, that is a DNA sequencer he is holding in his fingers.

ONT have commercialised DNA 'strand sequencing', not the exonuclease method Illumina licensed for $18M.

The sequencing chip: ONT are not using any published chemistry in their system. The chips apparently have an ASIC core with a membrane covering them supporting the nanopores. The aim is to release chips with 4000 or 8000 pores each. The enzymes used in the sequencing run at 20-1000bp/sec. Currently ONT are only using the change in current to differentiate bases, dwell time has been proposed as a way increase discriminatory power and sequence MeC, etc.

The sequencing is done by reading 3bp Kmers, i.e. the pore is large enough to hold three bases inside. These three bases give a characteristic signature and as the strand translocates one base at a time sequential Kmers allow discrimination of the sequence.

Sample prep: The current preferred sample prep is to add a hairpin to the molecule to allow sequencing in both directions. Load the first strand, run through the hairpin and start reading the second strand all in one contiguous read. DNA can be fed through 3’-5’ or 5’-3’.

The chips are very stable and can be loaded with blood to allow sequencing of DNA in solution with no sample prep at all. Clive is going to get a reputation as a mean b*****d after describing sending a vegetarian colleague to an abattoir to collect blood, then getting it from someone’s pet rabbit and finally sending another ONT employee into a river to collect raw sewage. All in the name of progress ;-)

Genome sequencing: Clive discussed a couple of projects. First ONT have sequenced PhiX. But they did it in a single contiguous read. First pass sequencing showed about a 4% error rate but the hairpin double-strand sequencing reduced this to 0.1-2% and the errors are in know locations of the genome as some Kmers are know to be “wobbly”. Next they sequence Lambda, all 42Kb in single reads, and as 5 and 10Kb fragment libraries. The Lambda sequence was done as a 100kb read, 42kb from each direction. The most interesting thin here was that the quality of the 1st, 10000th and 40000th bases is the same. Unlike all other systems that show some form of decay in quality, ONT may be able to give us almost infinite read length, whole chromosomes perhaps?

RNA-seq and Epigenetic modification: MeC and hMeC have been looked at but many other could potentially be analysed.

Clive briefly mentioned RNA-seq with no cDNA conversion but immediately pointed out thsat though this had been demonstrated there were no plans to commercialise the application at this time. An interesting feature about the ONT methodology is that once one strand has been sequenced a new one can load in and begin sequencing. I can imagine this is going to allow some really cool RNA-seq once they finish the development of that, even with only 4000 pores per chip, at 1000bp/sec you could sequence nearly 3M transcripts of 5Kb average length. With no cDNA intermediary!

GridION: The GridION was presented in a 20-node cluster format that, when loaded with 8,000 pore chips, could sequence a human genome in just 15 minutes. The GridION will be available first with 2000 pore chips, 4000 and 8000 will come in the mid-term. Using the “Run until” technology, a user can specify how much data they want form a samples and leave the instrument to run for just long enough to get all the data. I did not get a chance to see the pricing

The star of the show - MinION: This is a disposable USB sequencer that will generate up to 1Gb of sequence in the field. Just plug into your laptop and sequence! The chips are capable of 5-25Gbp per day but runs are limited to 6 hours due to chemistry.

The possibility of sequencing in the field brings a whole new dimension to disease and agricultural research. Being able to swap a patient or collect a fungal sample and sequence to identify which drug or fungicide should be used is going to revolutionise research, health and agriculture.

We can't place an order just yet. I certainly hope to start some early access collaborations (hint, hint) and am still not really sure what difference this is going to make to the world.

PS: sorry if there is anything incorrect in this post, there was a lot in Clives talk and I have been grabbing a few minutes here an there to get the post together.

Oxford Nanopore confirmed at AGBT

ONT will be speaking at AGBT. 11:40 am-12:00 pm

Clive Brown, their Chief Technology Officer will be talking about “Single Molecule ‘Strand’ Sequencing Using Protein Nanopores and Scalable Electronic Devices” just before lunch on Friday at 11:40. But only for 20 minutes.

Back in April last year Clive posted on SEQanswers "What if your reads were over 100kb, very accurate and you had a mountain of them?"

See you there.

PS: Illumina licensed ONTs exonuclease sequencing methods only, and I am not sure what deal they have on the strand sequencing methods. Either exonuclease is going to come out in a blast of publicity later in the year or Illumina will need to negotiate again over strand sequencing.

Nanopore sequencing: is the hype about to end?

A follow-up post to this one explains what nanopores are and how they can be used for DNA sequencing.

There has been a lot of hype around Nanopore sequencing for a number of years. The promise of very long single molecule sequencing with nucleotide modifications being directly read out is the holy grail of sequencing technology. The fact is that it has been hard to translate a relatively simple concept into reality.

For the past three years attendees at AGBT have been waiting for Oxford Nanopore to speak. To date they have been very reluctant to say much publicly and although it has not been confirmed, the rumour mill is hot with speculation that this year is finally the year that they will talk.

But what will they talk about and is the Next Next-Gen (N2GS) just around the corner?

What am I hoping to hear about: I am hoping for a genome, PhiX would be OK (any nanopore sequence would be better than nothing), but I’d admit to being disappointed if this was all. It would be great to see a complex genome presented, Yeast or C. elegans. Of course what I really hope is that they will talk about their sequencing of a Human genome and if I allow my imagination to run away then I am looking forward to details of a long-read single molecule 1000 genome project.

What do Oxford Nanopore offer: ONT are developing nanopore-based technologies, the most interesting of which for this post is DNA sequencing.

ONT’s sequencing that has been discussed publicly uses an α-haemolysin pore coupled with an exonuclease. Rather than feeding an intact DNA strand through the pore and reading out the bases, the exonuclease-sequencing approach cleaves each base from the end of the DNA strand, those bases translocate through the pore and are detected and the DNA seqeunce is read out. Many thousands of these nanopores are required to run in parallel to sequence a genome. And part of the parellelisation comes in the form of the GridION system, where multiple sequencing chips can be run together.

Strand-sequencing has not been dropped by ONT though and Hagan Bayley has modified the pore to improve discrimination of bases. Others have also demonstrated methods to slow down the translocation of DNA through the pore by coupling it to a polymerase, which ‘ratchets’ the samples through a base at a time.

The GridION system was ‘reviewed’ in a post over at Genomes Unzipped by Luke Jostins almost exactly one year ago. And whilst this post has scant details on the sequencing to be fair very little additional has been revealed in the following 12 months. Lukes post does describe the compute-cluster-like architecture that ONT have developed to house future sequencers. A user could buy one or one thousand and do as much or as little sequencing as they need. What the impact will be on service providers like BGI will be interesting to see.

“Run until” technology: ONT’s exonuclease or strand-sequencing approaches, packaged in the GridION format, will allow scientists to load the instrument and continue to sequence until ‘enough’ data has been generated. How much is enough is determined by the user ahead of the run and the real-time analysis and monitoring will mean only the required data are generated.

It is theoretically possible to make use of this approach, or something similar today. By running highly multiplexed samples and performing analysis between runs it would be possible to generate almost exactly the required depth of sequencing per sample. Once a sample has been shown to be of high quality then only the number of indexed reads is required to calculate how much more sequencing is needed. This could be implemented on the other sequencing platforms, but I suspect is too difficult to implement in most labs today. Even if the efficiency gains could be worthwhile.

ONT and Illumina: In 2009 Illumina invested $18M in ONT and bought an exclusive license to "market, sell, distribute, and service BASE( BAyley SEquencing) technology.What this means for ONT and Illumina today is less clear and I am not certain if other milestone payments have been paid. Illumina certainly want to stay ahead of the competition (LifeTech, Complete and Roche). ONT might be their not-so-secret weapon.

What does the future hold: Dr Sanghera (ONT CEO) was quoted saying that the $1,000 genome will be possible “within three to five years” in a March2011 interview with The Economist. Things have sped up faster than ever in the last year and the $1000 genome looks like it is already here. Can ONT deliver us the $100 or $10 genome? I am certainly looking forward to having them piling the heat on Illumina and Life Tech.

Lastly, back in 2009 when Clive Brown ONT CTO) was interviewed in BioIT World he said "before launching a product, you have to run it in house for months, doing genome-centre type things". Hopefully we are about to find out exactly what those genome-centre type things are.

How does a nanopore sequencer work?

You may have come here from my other Nanopore post, if not then this is a follow-up to that and is meant to outline Nanopore sequencing technologies.

How does a nanopore sequencer work: A nanopore is a very small hole, the generally under 1nM in width in a membrane of some kind. It can be made from a biological molecule or ‘punched’ into a solid surface using an electron beam. Nanopore sequencing has a very simple basic principle, DNA strands or single nucleotides are driven through a nanopore electrophoretically. As each nucleobase passes through the pore the current is affected and this change allows sequence to be read out. Each base has a characteristic change in current and, perhaps just as importantly a specific dwell time in the pore. One of the first publications of the idea was from George Church in a 1995 patent (Church et al), this was a year or two before Shankar Balasubramanian and David Klenerman invented the SBS chemistry and formed Solexa. Nanopore sequencing has been around as an idea for a while!

The earliest demonstrations of the technology used α-haemolysin or Mycobacterial porin A (MspA) biological nanopores. Biological and solid-state pores have now been demonstrated and hybrid systems have also been discussed.

Biological nanopores: cells are very good at making biological nanopores like α-haemolysin, they also make many other similar molecules and it is possible to design pores with specific characteristics using site-directed mutagenesis. These can be checked at the atomic level with X-ray crystallography to verify their structure. α-haemolysin pores have been widely used as the hole in the middle is only wide enough for single-stranded DNA to pass through. Unfortunately DNA moves through these pores very rapidly making detection of each base almost impossible. Slowing its translocation down has been a goal of nanopore research. DNA can be coupled with enzymes like DNA polymerase and “ratcheted” through the pore. Oxford Nanopore have been working on “sequencing-by-digestions” where an exonuclease sits above the pore cleaving individual bases from a strand of DNA which pass through the pore allowing sequence to be read out. Biological pores also offer the promise of detecting not just DNA sequence but also interacting DNA:protein molecules and possibly protein sequence. A big challenge for biological nanopores is that they are often embedded in fragile lipid bi-layers and can be affected by physical conditions such as temperature, pH, etc. It can also be difficult to make large arrays of nanopores and we would need many thousands of pores to sequence a Human genome.

Solid-state nanopores: Many groups are working on solid-state systems using ion or electron-beam sculpting of pores in Silicon nitride membranes. Making the pores and the membranes is challenging as spacing and thickness need to be carefully controlled but these systems are much less affected by physical conditions and may also be coupled to electronic or optical read-out systems. The latest advancement appears to be the use of graphene, a two dimensional sheet of carbon atoms as the membrane of choice. The use of solid-state nanopores is not as advanced as the biological approach and similar challenges for controlling the speed of DNA translocation are as yet unmet.

How will my sequencing be affected: Nanopores offer the promise of sequencing a base per millisecond with high accuracy and detection of base modifications like methyl-C. At these speeds one million base pairs can be read-out in about 20 minutes. With 1000 pores in an array a Human genome might be sequenced in an hour or so.

Nanopores also offer a major advantage over current methods even if speeds don’t quite approach this, no labelling is required and single molecules are analysed. This means there are few reagents and possibly no sample prep other than extracting DNA, so it should be easier and cheaper than current methods!

Church et al. Characterization of individual polymer molecules based on monomer-interface interactions. US patent 5,795,782 (1995).
Venkatesan and Bashir. Nanopore sensors for nucleic acid analysis. Nature NanoTech 2011
Branton et al. The potential and challenges of nanopore sequencing. Nat Biotechnol. 2008