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  • #31
    Originally posted by RALeski52 View Post
    My experience has shown me that both technologies are important to provide optimal assemblies, especially when you work in novel environments where reference genomes are lacking.
    I'll second that. At the moment, no single technology is free of flaws. By combining two of them, you get rid of almost all problems. 454 and Illumina happen to be the cheapest solution at the moment for de-novo at really high quality (meaning for me <= 1 error per 1 megabase in non-repetitive areas): the 454 homopolymer problem is canceled out by Illumina and the Illumina GGCxG problem by 454. And the variation in coverage of each technology is balanced out by the other technology. Just perfect.

    Now, whether 454 can stay as partner in hybrid solutions is another question. Things will get very interesting if Ion gets to 200 - 300 bp reads or if PacBio gets to an accuracy >= 93-95%.

    Until then: hybrid for me means 454 + Illumina

    B.

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    • #32
      they've had the 1K upgrade at the CGR liverpool for almost a year now. Last I heard the average read length was still under 600 in their hands but this may have improved recently

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      • #33
        From a core facility/service provider perspective, with both 454 and HiSeq: we see a dramatic drop in 454 projects other than amplicon. A lot os this is 16S. Here are some pie charts to prove the point. We don't like this trend as these samples cause much more trouble in the lab than 'simple' genomic DNA samples, and they require custom base-calling analyses.

        We heard some centers working on getting 16S amplicon projects running on the HiSeq. IMHO Roche/454 need to come up with cheap (!) nice long (!) reads soon (!) in order not to get pushed back to a very small niche...

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        • #34
          Around the same time 1kb reads were being mentioned (2009), Roche were also talking about reducing the capture bead and PTP well sizes in order to generate more reads per run. Has anyone heard anything more on this? I think this would be more advantageous than the increased read length for may applications.

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          • #35
            Originally posted by TonyBrooks View Post
            Around the same time 1kb reads were being mentioned (2009), Roche were also talking about reducing the capture bead and PTP well sizes in order to generate more reads per run. Has anyone heard anything more on this? I think this would be more advantageous than the increased read length for may applications.
            I believe the instrument issue there is the camera.

            But it makes you think. Would a complete retread of the 454 be competitive with Illumina? Say with 1 um beads. 400x more reads/run. 400 million 800 base reads would be 320 billion bases of sequence.

            Well, that would put it in the same weight class as a HiSeq.

            --
            Phillip

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            • #36
              Originally posted by TonyBrooks View Post
              Around the same time 1kb reads were being mentioned (2009), Roche were also talking about reducing the capture bead and PTP well sizes in order to generate more reads per run. Has anyone heard anything more on this? I think this would be more advantageous than the increased read length for may applications.
              Isn't this the Titanium upgrade?

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              • #37
                Certainly camera technology has improved over the past few years to where an upgrade or new model could have a higher resolution/more sensitivity or whatever it would require to image more, smaller wells. Am I wrong to think this?

                Also, they really really really would need to automate as much of the emPCR process as possible. If Life Tech can do it for Solid and PGM, why hasn't Roche/454 bothered with it? I haven't used a REMe, but why when developing it did they not just make a little box that can do the whole breaking, enrichment, recovery process instead of a device that requires a separate liquid handling system and that only does a part of the process??

                And I've also wondered recently why they never worked to automate the library prep? Their MagNA Pure systems remind me of the SpriWorks so perhaps it would've been possible for them to come up w/ kits and a method card to automate the prep. Probably way too late now for this to be of much use though.

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                • #38
                  Originally posted by RCJK View Post
                  Certainly camera technology has improved over the past few years to where an upgrade or new model could have a higher resolution/more sensitivity or whatever it would require to image more, smaller wells. Am I wrong to think this?

                  Also, they really really really would need to automate as much of the emPCR process as possible. If Life Tech can do it for Solid and PGM, why hasn't Roche/454 bothered with it? I haven't used a REMe, but why when developing it did they not just make a little box that can do the whole breaking, enrichment, recovery process instead of a device that requires a separate liquid handling system and that only does a part of the process??

                  And I've also wondered recently why they never worked to automate the library prep? Their MagNA Pure systems remind me of the SpriWorks so perhaps it would've been possible for them to come up w/ kits and a method card to automate the prep. Probably way too late now for this to be of much use though.
                  The reduced bead sizes were mentioned in the Lisbon EMEA meeting in June 2009. This was post Titanium release which came in around the end of 2008, beginning of 2009. I vaguely remember they wanted to reduce everything by 50%, giving 4X more reads per run. I think the problems are probably stemming from producing the PTP. I can imagine those are quite tricky to produce.

                  Personally, I don't have too many issues with the 454 library prep protcol. It's much easier than the Illumina one - no gel cut as standard, no PCR enrichment only 2 clean-ups. I don't think there's too much call for that being automated unless you wanted massive throughput. In that case, you it should be fairly automatable on most flatbeds.

                  However, I would welcome a "magic box" that does the breaking, enrichment and recovery though. Those steps are a pain in the you-know-what.

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