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  • #16
    The press releases are out!

    Sorry, this page has been moved or deleted Are you looking for any of the following? Pu

    Oxford Nanopore's AGBT presentation should have just finished up, so the embargo is off.  Oxford was kind enough to chat with me last night ...

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    • #17
      Originally posted by maubp View Post
      The press releases are out!

      Sorry, this page has been moved or deleted Are you looking for any of the following? Pu

      Oxford Nanopore's AGBT presentation should have just finished up, so the embargo is off.  Oxford was kind enough to chat with me last night ...

      http://pathogenomics.bham.ac.uk/blog...w-for-this-blo
      So, is there real human data yet, or are we to extrapolate on lamda phage data?
      Last edited by Geneus; 02-17-2012, 10:25 AM.

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      • #18
        Patience Real data should be coming ... soon we all hope.

        Is ONT public or privately held? I am lazy to look it up.

        Originally posted by Geneus View Post
        So, is there real human data yet, or are we to extrapolate on lamda phage data?

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        • #19
          Originally posted by GenoMax View Post
          Patience Real data should be coming ... soon we all hope.

          Is ONT public or privately held? I am lazy to look it up.
          Oxford Nanopore’s shareholders include IP Group Plc, which owns 21.5 percent, hedge-fund manager Lansdowne Partners LP and Invesco Perpetual, the U.K. group of mutual funds. The company also has individual shareholders, including company managers, and employees have stock options.

          Source: http://www.businessweek.com/news/201...e-in-2012.html

          Comment


          • #20
            Well it was certainly worth waiting for, now when can I preorder some MinIons (though ignoring there marketing I'm going to be calling them minions! After all they're cheapish and disposable!)

            It would be good to see some human or eukaryote data!
            Last edited by aeonsim; 02-17-2012, 01:38 PM.

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            • #21
              definitely more useful than i was anticipating. I'm still unsure about a few parts. At least they are upfront about the raw error rate which wasn't too bad. The low read count is still concerning with such an error rate, but it makes a great companion for hybrid datasets.

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              • #22
                So let me run a score card on my own prognosticaltion

                Originally posted by BBoy View Post
                My predictions: long read lengths, on par or beyond what PacBio can achieve;
                Check, though the claimed read length is massively higher than Pac Bio

                Originally posted by BBoy View Post
                raw accuracy better than PacBio, but probably still in the low-mid 90s;
                Check check

                Originally posted by BBoy View Post
                poor data output per rack, with high consumable costs due to relatively low multiplexing per chip
                Yes and no. The raw cost per base is apparently low, but the multiplexing is indeed presently very low - hundreds to a few thousand wells. This is the main issue I see for them - with solid state technology being where it is they need to rely on biological pores to get the resolution and therefore the outlook for massive parallelism is somewhat murky.

                Overall impressive technical results, I need to disgest them some over the weekend. Seems more like a threat to the PGM than PacBio at first glance - simple sequencing at a low capital cost, with what seems to be much lower reagent consumption and simpler sample prep.

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                • #23
                  Originally posted by BBoy View Post
                  Define "easier". Easier to achieve higher raw accuracy... yes. Easier to achieve reasonable multiplexing... not only no, but hell no. At least not for as long as they are using biological nanopores.
                  Can't believe I missed this...tried to resist replying.

                  Multiplexing for PacBio requires real time multi-axis nanometer-scale alignment of multiple excitation lasers and emission collection equipment across a macroscopic (~4mm circle for 2x75,000) array of ZMWs that also has to be machined/synthesized optically flat. This is an optics problem that seems to shame the Hubble telescope.

                  Multiplexing of a biological nanopore array requires coating an array of sensors with biological-esque membrane and depositing one (and only one) pore atop each sensor (this is ultimately a statistics problem). I have seen data from nanopore companies that suggests this is (relatively) trivial. Then await the stream of voltage data and hope your pore can discriminate bases or groups of bases. Oh and you need a Transmeta processor and RAMBUS RAM too...!

                  In my mind (and perhaps slanted/naive perspective), there is no contest in the difficulty of methods. But difficulty is not much more than a badge of honor in this business, then again PacBio has a functional instrument and ONT has years of promises and two phases of vaporware launches.

                  Biological nanopores are a probably not a passing fad...likely the world will need many years of fab technique development before a solid-state nanopore can replicate the precision of mspA, aHL, or whatever secret pore ONT is using (likely a mutant of mspA). As far as I know there is no angstrom/atom level 3D silicon fabrication technique out there that works in huge parallel.

                  Again...I (perhaps obviously) have limited knowledge of anything other than molecular biology. I could be very far off with the above...but it sure is fun to speculate about!

                  Bottom line in my book...I have _never_ been blown away by a sequencing announcement. Everything ends up being hype followed by incremental increases in [RL/Tput/Accuracy/sampleprepsimplicity].

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                  • #24
                    Originally posted by ECO View Post
                    Multiplexing for PacBio requires real time multi-axis nanometer-scale alignment of multiple excitation lasers and emission collection equipment across a macroscopic (~4mm circle for 2x75,000) array of ZMWs that also has to be machined/synthesized optically flat. This is an optics problem that seems to shame the Hubble telescope.
                    Yes, this was an unfortunate design decision in their instrument design that is relatively easily correctable. Fabricating an integrated waveguide distribution system that delivers light to the ZMWs and is locally aligned with sub-um precision is a trivial task by today's technology standards - every microprocessor today does that for billions of features with a precision of a few nanometers. Slapping a dedicated disposable CMOS active pixel as the local sensing element is also a well understood task. None of these are conceptual limitations. Whether Pac Bio gets to address them before they run out of cash is an open question.

                    Originally posted by ECO View Post
                    Multiplexing of a biological nanopore array requires coating an array of sensors with biological-esque membrane and depositing one (and only one) pore atop each sensor (this is ultimately a statistics problem). I have seen data from nanopore companies that suggests this is (relatively) trivial. Then await the stream of voltage data and hope your pore can discriminate bases or groups of bases.
                    Agreed. But how do you pack millions of these on a sub-1um pitch in an ordered array that is aligned to the sensor underneath? Please don't tell me "directed self assembly"

                    Originally posted by ECO View Post
                    In my mind (and perhaps slanted/naive perspective), there is no contest in the difficulty of methods.
                    And neither is there one in my mind. The technology that solves PacBio's problem was developed decades ago and is in massive use today (every cell phone camera basically implements it). The technology required to create an ordered array of millions of biological nanopores exists only in Nature papers :-)

                    Originally posted by ECO View Post
                    Biological nanopores are a probably not a passing fad...likely the world will need many years of fab technique development before a solid-state nanopore can replicate the precision of mspA, aHL, or whatever secret pore ONT is using (likely a mutant of mspA). As far as I know there is no angstrom/atom level 3D silicon fabrication technique out there that works in huge parallel.
                    Hey, people are publishing papers on graphene for nanopore sensing. Talk about putting lipstick on a pig. Yes, synthetic nanopores are with the required dimensions and tolerance are at least a decade away. Biological nanopores are here to stay.


                    Originally posted by ECO View Post
                    Again...I (perhaps obviously) have limited knowledge of anything other than molecular biology. I could be very far off with the above...but it sure is fun to speculate about!
                    And I have limited knowledge of anything other than semiconductors. So we have a deaf communicating with a mute and a blind (now THAT was a funny movie back in its day :-)
                    Last edited by BBoy; 02-18-2012, 02:14 AM.

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                    • #25
                      I noticed the video suggested the nanopores can be adapted for proteins. Does this mean there'll be a single-molecule protein sequencing pipeline available in the near future?

                      Sorry, this page has been moved or deleted Are you looking for any of the following? Pu

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                      • #26
                        My understanding from what they told me is that there isn't a 1:1 relationship between circuits and pores. In fact there are more many circuits than pores, and a pore can address between 0 and 4 circuits. Presumably they have a method for sorting out the cross-talk (sorry not to be more specific).

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                        • #27
                          Originally posted by BBoy View Post
                          Yes, this was an unfortunate design decision in their instrument design that is relatively easily correctable. Fabricating an integrated waveguide distribution system that delivers light to the ZMWs and is locally aligned with sub-um precision is a trivial task by today's technology standards - every microprocessor today does that for billions of features with a precision of a few nanometers. Slapping a dedicated disposable CMOS active pixel as the local sensing element is also a well understood task. None of these are conceptual limitations. Whether Pac Bio gets to address them before they run out of cash is an open question.



                          Agreed. But how do you pack millions of these on a sub-1um pitch in an ordered array that is aligned to the sensor underneath? Please don't tell me "directed self assembly"


                          And neither is there one in my mind. The technology that solves PacBio's problem was developed decades ago and is in massive use today (every cell phone camera basically implements it). The technology required to create an ordered array of millions of biological nanopores exists only in Nature papers :-)


                          Hey, people are publishing papers on graphene for nanopore sensing. Talk about putting lipstick on a pig. Yes, synthetic nanopores are with the required dimensions and tolerance are at least a decade away. Biological nanopores are here to stay.



                          And I have limited knowledge of anything other than semiconductors. So we have a deaf communicating with a mute and a blind (now THAT was a funny movie back in its day :-)

                          Ouch...this stuff is waaaaaay over my head. I'll need to ask my brother to read and decipher (he's Chief Systems Technologist at a large chip foundry). Who knows, maybe I can peak his interest.

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                          • #28
                            Originally posted by BBoy View Post
                            Yes, this was an unfortunate design decision in their instrument design that is relatively easily correctable. [snip]

                            And I have limited knowledge of anything other than semiconductors. So we have a deaf communicating with a mute and a blind (now THAT was a funny movie back in its day :-)
                            Awesome post. I can't say anymore about waveguide distribution...this would be a fun conversation over a beer.

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                            • #29
                              The first thing I would want to do would be to gather 3x coverage of 50kb+ reads from a moderate to large eukaryotic genome. (1 gigabase +). Preferably something already considered "sequenced". For example, maize, soybean or some mammal. I am hazy on the costs of doing this on a GridIon, but for soybean on a few MinIons it looks like it would cost about $3000. If there do not seem to be any major issues I would think most of the thorny issues involved in de novo genome sequencing disappear.

                              1 Gbp genome -- go 50x with Illumina PE reads (maybe 3 HiSeq lanes). Don't bother with the ME reads, making those libraries takes real effort. Then assemble on a scaffold of (Min|Grid)Ion reads (50 - 100 kb, right?). Instant chromosome sequences? Maybe you still have some issues with centromeric heterochromatin. But with 100 kb reads, maybe not. That is 1 month project you can do for $20K, right?

                              So I am reading right that they are only releasing phage lambda data though? Or are they even releasing that? My experience is that even E. coli Dam methylation is incomplete in lambda clones. Anyone seeing an increase in errors at Dam (GATC) sites?

                              Another potential issue: it has never been clear to me what level of damage exists in a typical DNA prep. Seems like oxidation/adduct/apurinic/pyrimidine-dimer sites might be quite common and we would not notice it using normal (colligative) molecular techniques.

                              Even if this were the case, passaging the DNA of our species to be de novo sequenced through a non-modifying host would probably only double the cost of a new genome sequence. (Eg, make a cosmid library in a modification minus host strain.) Or, if you just end up with an "N" at the position of a modified or damaged base -- probably could live with that.

                              --
                              Phillip

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                              • #30
                                Originally posted by pmiguel View Post
                                Another potential issue: it has never been clear to me what level of damage exists in a typical DNA prep. Seems like oxidation/adduct/apurinic/pyrimidine-dimer sites might be quite common and we would not notice it using normal (colligative) molecular techniques.
                                This was an interesting problem for sample prep at PacBio, as we used several exonucleases to remove non-covalently closed SMRTbells (which also cut at abasic sites).

                                We encountered significant internal damage (exo-labile abasic sites) with inserts over a certain size from "high quality" DNA preps, and initially couldn't make any products from larger PCR products. I interpreted this as there were >1 abasic sites in every insert molecule larger than a certain size.

                                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.

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