[pmiguel]

Just one of the many fun challenges of single molecule sample prep. Hopefully Oxford can already cope with real biological abasic sites (not stable THF synthetic ones) and or the adducts you mention. Basically adds additional moieties they need to detect.

Seems like studies looking for oxidation products (8-oxo-G) always find it. Who knows how many methylation/hydroxymethylation/etc. variants are present in natural DNA samples.

Does the commercial release schedule Oxford plans seem realistic to you? I can't find any data sets they have released. So commercial release of MinIons and GridIons later this year seems fantastically aggressive to me.

In Sequence is quoting Oxford Nanopore's price per gigabase for the GridIon at around $40. $40,000/ terabase is Illumina HiSeq2500 territory 6 months from now. At that price point Illumina gives you 1-2 orders of magnitude lower error rates and that's it. Every other possible advantage would be with the GridIon.

Of course we already know the limitations and strengths of a HiSeq, whereas we have only Oxford Nanopore's marketing hype to make an assessment of their product.

Thing is, I always assumed the final state of sequencing would be a 4th gen machine that does no chemistry on the templates it sequences. That is, it would be a DNA reader. But that eventuality was over the "horizon". Might have taken us another decade to get there.

--
Phillip

[gringer]

Thing is, I always assumed the final state of sequencing would be a 4th gen machine that does no chemistry on the templates it sequences. That is, it would be a DNA reader. But that eventuality was over the "horizon". Might have taken us another decade to get there.

There are still a few more opportunities for additional generations of sequencing technology.

Consider that the sequencing still needs a lysis stage. What about in-vitro / in-vivo sequencing, or completely non-invasive sequencing?

[pmiguel]

There are still a few more opportunities for additional generations of sequencing technology.

Consider that the sequencing still needs a lysis stage. What about in-vitro / in-vivo sequencing, or completely non-invasive sequencing?

And plenty of work to do in the single molecule sampling in lysed samples arena. The devil is in the details.

I would say a completely non-biological sensor (solid state) would be worthy of the appellation "5th generation".

--
Phillip

[BBoy]

Seems that Rothberg shares my concerns about scaleup

http://www.forbes.com/sites/matthewherper/2012/02/18/who-doubts-the-usb-thumb-drive-sequencer-a-rival/

True, he is not exactly unbiased, so this should be taken with a rock of salt.

[Pongo_T]

For the more technically minded: did Clive Brown show any primary data in the sense of simultaneously acquired nanopore current traces (there is none on the webpage, is there?) It is one thing to show that (1) strand sequencing basically works on short genomes and that (2) you have a mulitplexed nanopore chip which might be good for longer ones, but quite another to show that strand sequencing has actually successfully been done using that chip. There are lots of things where they must have made tremendous progress, not least the on-chip multiplexed 512-channel (for the Minion) voltage-clamp amplifier (CMOS-based, I understand) which has to have pretty low noise, a.s.f.

[pmiguel]

Seems that Rothberg shares my concerns about scaleup

http://www.forbes.com/sites/matthewherper/2012/02/18/who-doubts-the-usb-thumb-drive-sequencer-a-rival/

True, he is not exactly unbiased, so this should be taken with a rock of salt.

Strange, does Rothberg think the genome size of phage lambda is 5000 bases? 5x10^3 to 6x10^9 that would be six orders of magnitude. Lambda is about 48,500 bp.

Okay, I am being pedantic. It does not change his main point. Whether 5 orders of magnitude or 6 that does seem like a lot of ground to cover in less than a year. Why not sequence a bacterial genome? Or yeast? Would be really nice to see the sequence reads from a data set.

Guess we will be seeing...

--
Phillip

[Wallysb01]

How many times have we seen this?

Lets see some real data from a Eukaryote before we pass any kind of judgement. Heck any actual data would be a nice first step.

Its all marketing and spin until then...

[aeonsim]

How many times have we seen this?

Lets see some real data from a Eukaryote before we pass any kind of judgement. Heck any actual data would be a nice first step.

Its all marketing and spin until then...

Yes as promising as it looks we certainly need some real raw data, a lambda dataset and a Eukaryote would do much to to inspire confidence!

Though as soon as they make MinION preorders available we'll order half a dozen for testing just to get a feel for the data with our organism, and current methods. The minions really are an interesting looking product and have awesome potential as marketing tool that makes a nice profit.

If data from the minions looks good then the primary instrument becomes very tempting assuming a reasonable cost.

[Wallysb01]

Yes as promising as it looks we certainly need some real raw data, a lambda dataset and a Eukaryote would do much to to inspire confidence!

Though as soon as they make MinION preorders available we'll order half a dozen for testing just to get a feel for the data with our organism, and current methods. The minions really are an interesting looking product and have awesome potential as marketing tool that makes a nice profit.

If data from the minions looks good then the primary instrument becomes very tempting assuming a reasonable cost.

It certain looks cool. Sequence a genome in the palm of your hand! Who isn't willing to at least try that? And if it actually works maybe those little vacuum cleaners from GATACA that sequence your DNA in a few seconds will be possible! But really, who's going to get paper print out of such a thing....

[ECO]

Though as soon as they make MinION preorders available

If they are available in 2012, I would love to arrange for a SEQanswers community beta test of 10 of them...even if I have to hit my savings account to pay for them.

Unfortunately beta testers these days are under lock-tight NDA's so they can't share their real experiences. Chance of the above happening? You decide.

[gringer]

Strange, does Rothberg think the genome size of phage lambda is 5000 bases? 5x10^3 to 6x10^9 that would be six orders of magnitude. Lambda is about 48,500 bp.

Okay, I am being pedantic. It does not change his main point. Whether 5 orders of magnitude or 6 that does seem like a lot of ground to cover in less than a year. Why not sequence a bacterial genome? Or yeast? Would be really nice to see the sequence reads from a data set.

The actual output of the sequencers was probably a little more than 100kb. That lambda run was sequenced on a MinION (i.e. the "smaller" sequencer), generating complete 100kb fragments from single pores:

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

The MinION has 512 pores, and runs can take up to 6 hours, so it would be conceivable that the run that produced this sequence could have produced 10-50Mb of sequence in six hours depending on how many active sites are assumed (i.e. 2 more orders of magnitude than what is suggested in your statement). The GridION system can do 2000 pores and run for a bit longer. Assuming that's a ten times longer run time with similar processing length / quality, you get another 40~100x quantity of generated sequence. The GridION system can be racked, so rack up 10 GridIONs together and it comes to the magic 5 orders of magnitude using the systems that have already been reported in the press releases.

The press release I linked to above suggests a 20-node GridION system with 8000 pores per cartridge is feasible, and "would be expected to deliver a complete human genome in 15 minutes", and this agrees mostly with the back-of-the-computer-screen calculations I've done here.

[Pongo_T]

That lambda run was sequenced on a MinION (i.e. the "smaller" sequencer)

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

Are your quite sure it was actually done on the Minion or on any chip array? Was this made clear in the talk?

[gringer]

Are your quite sure it was actually done on the Minion or on any chip array? Was this made clear in the talk?

Hmm, I think I combined a few too many things because it was in a press release that had "MinION" in the title. The important thing that I was trying to get across was that this was a sequence from a single pore, and the sequencers will have hundreds or thousands of pores on their surface.

If it is assumed that it was on a different system from the MinION (e.g. on a single isolated pore) with a run time in excess of ~30h, you're welcome to take an order of magnitude off my estimates (so still one more to go, assuming a somewhat pessimistic calculation).

[gringer]

If it is assumed that it was on a different system from the MinION (e.g. on a single isolated pore) with a run time in excess of ~30h, you're welcome to take an order of magnitude off my estimates (so still one more to go, assuming a somewhat pessimistic calculation).

Here's an article that mentions tens of gigabases of sequence per day on a single GridION node with 2000 pores:

http://www.azonano.com/news.aspx?newsID=24324

This works out to 5 megabases per pore per day (assuming full pore occupancy), or about 200kb per hour. At this rate, a 100kb sequence should complete in the 6-hour run time of a MinION (even if assuming a 1/10th read speed compared to the GridION cartridges), so I'm going to stick with my initial "plausible, based on press releases" thoughts.

[pmiguel]

The actual output of the sequencers was probably a little more than 100kb. That lambda run was sequenced on a MinION (i.e. the "smaller" sequencer), generating complete 100kb fragments from single pores:

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

The MinION has 512 pores, and runs can take up to 6 hours, so it would be conceivable that the run that produced this sequence could have produced 10-50Mb of sequence in six hours depending on how many active sites are assumed (i.e. 2 more orders of magnitude than what is suggested in your statement).

That is exactly the point. If the MinION is capable of such a feat, then why is the only report of its sequencing DNA that of single read lambda genomes? (And where are the reads generated during the run?) Why not knock off E. coli in 6 hours? Use a dam- dcm- strain if methylation is a problem initially. Also, why not release the sequence reads? If a human genome can be sequenced on a score of GridIONs in 15 minutes, why not do that?

But since our reaction has been one of nearly blanket acceptance, it looks like Oxford Nanopore made the right call. Why release data when nearly everyone will accept what you say without question?

If we were talking about a instrument a couple of years from commercial release, then I would say our attitude of polite astonishment would be warranted. But later this year?

I think two extreme possibilities include:

(1) Everything is exactly the way it is portrayed in the press releases. No data release because they know they will have real instruments available for sale in 6 months and they will sell every single one they make.

(2) There has been some sort of disconnect between what is being marketed and what is actually possible. No data is being released, because there is nothing to release.

Seems like our initial reaction was to presume possibility 1. Rothberg presumes something like 2. I don't see personally see any information that makes it possible to distinguish between the two possibilities. You can claim "sour grapes" all you want about Rothberg's comments. But at this point his claims are backed by the same amount of data that has been publicly released as Oxford Nanopore's claims: zero.

I am just wondering though, did anyone at the presentation at AGBT ask for the read data from the lambda genome sequencing?

I cannot imagine that I would have at that juncture. PacBio had been trumpeting their potential for years by the time they were 6 months from a commercial release. By that time the questions about their capabilities had become pointed. My impression is that Oxford Nanopore has taken a much more low key approach until now. We have all seen amazing events unfold in our field over the last 5 years. Nearly anything seems possible. Still, we should ask for data before we allow ourselves to become part of the hype. Has anyone?

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Phillip