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  • 454 future

    It just happen to me the other day - why 454 is stuck with the design put forth by Rothberg eons ago? It has so many limitations that essentially kill any possibility for its further development. Correct me if I am wrong, but 454 still has the longest read capability that is not matched by any other platform, at least reliably. I think it is a great advantage of the platform and it is a shame that Roche fails to bank on this pushing further development much more agrressively.

    The major limitation seems the platform throughput, which is essentially cast in stone (glass) because it is directly connected to the geometry of PTP, primarily to the linear density of reactors. But why it cannot be increased? If Ion Torrent guys can pack much more those reactors on a much smaller chip, why 454 cannot? GS chunior plate is about the same size and protocols are substantially simpler than for the big boy, so why not take this boy and make it competitive?

    Has it something to do with optical interference for signal detection? I am not expert here at all, but I thought that CCD with a much higher resolution can be deployed. As result of further miniaturization, Ion Torrent can use tiny beads for its chips, which enabled automatization of emPCR by running it in a pouch. Going this way eventually limits read length, though, since tiny bubbles in emPCR can hold only limited amount of reagents, so amplification of long fragments becomes problematic. But a golden middle can be found, I am sure. The current method uses roughly 30 micron beads, while Ion Torrent uses something in micron size, so there is a lot of room for improvement. Yet, just 10-fold increase in reactor density will put 454 right in the throughput range of most popular platforms, 3.5 Gb to 35 Gb, but with undisputable advantage of long reads. What dou you, guys, think?

  • #2
    I've also thought of some of the same things with regards to 454's platforms, and would therefore assume that the folks at 454 have looked into some of the points you raised as well. I'm not an engineer or anything, so I have no idea whether some of these things would be possible, but it doesn't seem like it would be such a great leap. Surely the technologies in the instruments have improved in the past few years so that an upgraded version could've been released.
    I really don't know why they haven't upgraded the Jr to be able to do the XL+ length reads. I think that would've been that instrument's only shot at being viable. Also, they really really should've invested more in automating the prep, especially the emPCR portion of the process. Lowering their prices would've helped too.
    I'm sure a big part of all of this is being a part of Roche and the support/lack of support they get from higher up within the company. As was mentioned in another thread, why not invest some of the $5.7B into 454? I'm sure even a small part of that would be a big boost to the R&D dept.

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    • #3
      The resolution of the camera is certainly a limiting factor. As well as I can figure, the camera produces 16 megapixel images (older .pif images are 32 MB each, and according to the GS FLX software manual, data is stored at 2 bytes per pixel in those images, so therefore 16 megapixel images). There are about 1.6 million wells on the plate, so that's about 10 pixels per well. Curious about how the pixels and wells line up, I zoomed way in on an image. Here's what I saw:

      You can see that each well is represented by a block of about 9 pixels. Where there are beads in adjacent wells, there is some overlap. You might be able to shrink it so that each well is represented by a block of 4 pixels, but I doubt that it could be shrunk any more and still get reliable data, unless you could reproducibly line up the camera accurately with the plate every time. It seems to me that the only way to get significantly more data is to design a new instrument that either has a higher resolution camera, or a different sensor technology, perhaps something like Ion Torrent's pH method. Last I heard, they are working on something like that as well as a solid state nanopore sequencing technology in collaboration with IBM. I don't know if we'll ever see such products, but they were working on them recently and as far as I know they still are. I do think, however, that the FLX is probably the best we can expect from the current technology.

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      • #4
        Folks really need to walk on the private sector side of public companies. There is no way on earth that any company could handle or manage 1 billion in R&D...for science tools, let alone 4 or 5. The sad reality is that most of the profit from the big three (or at least from 2 of the big 3) has come from drastic cuts in both staff and expenses. When these fall, there is no rationnal, there are only spreadsheets and MBAs. Most companies now work with flawed Harvard business model of M&A followed by integration, followed by massive reduction in costs with a certain viable time to hold on to IP and profit. Then all craps out until the next cycle... I am sure 454 is interested in all this stuff but there is no way they can implement. Implementation is a huge issue, again your typical analysis of 6 sigma lean and black belt process's are put on paper for ages, powerpoint presentations, endless meetings. These days, laying low in R&D seems to be more secure than rocking the boat...

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        • #5
          No reason why we as end-users should subjugate what would be best for us to what investors think is best for them.

          One issue here has to be employee morale -- how would you feel working for Roche Applied Sciences, knowing your parent company is attempting to purchase your main competitor while your resources dry up?

          --
          Phillip

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          • #6
            But then it is sickening to see that Roche can find folds more billions to make offer for takeover of Illumina, yet hesitates to invest a fraction of it in picking up an existing perspective model for aggressive development. It seems that progress has the only way of starting in a small group of skinny smarties with lean brains, move ahead for a while fueled largely by enthusiasm until it gets noticed by fat coffers just to come to a screeching halt. Whatever economy model, it does not seem as capitalism anymore.

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            • #7
              Sure it is, it is the capitalism that most life tool companies work buy. Innovation is actually quite slow and low, volume acquisition of small innovation is how things work now... Frankly, I am sure no one is suprised by Roche massive acquisition. It's not about what people can do, it's about share pices and how the CEO is bonified, the rest (including sickness and disease) is rather remote... Let's not be naive folks.

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              • #8
                No, this is not capitalism anymore. I guess we need another Carl Marx to define what is now. If that was back 100 years ago, we would not have Henry Ford or giants alike, which shaped tphe world we have now. Something is wrong now.

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                • #9
                  Well Roche has announced some future upgrades to the Jr. platform including longer reads and automated library prep & emPCR. It's about time they've realised their platforms will need to be more automated to be somewhat competitive. Hopefully the automations can be used for the FLX+ as well and that the emPCR automation will be for the entire workflow and not just a miniature version of the REMe.

                  http://www.genomeweb.com/sequencing/...ades-gs-junior

                  A new software package and protocols for paired-end sequencing to enable multiplexing are available immediately. Longer read lengths and automation of the library preparation and the emulsion PCR workflow will be available in late 2012 or early 2013.

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