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  • #16
    BTW, roche at 1% growth last quarter are clearly struggling.

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    • #17
      Then I'd be dreaming

      Originally posted by clivey View Post
      What if

      Your reads were over 100kb, very accurate and you had a mountain of them ?
      In this scenario would I also be cruising down the Pacific Coast Highway in a Ferrari with Elle McPherson in the passenger seat?

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      • #18

        What applications do you need 1000bp or more for that can't be broken down into short uniquely barcoded amplicons tiled across regions?

        That's what makes our jobs so much fun!
        surely the inverse of this statement is also true. What applications do you need tiled short reads for that cannot be addressed with long reads (of sufficient qual).

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        • #19
          Originally posted by clivey View Post
          Your reads were over 100kb, very accurate and you had a mountain of them ?
          Are you an agent of a Nanopore company?

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          • #20
            Longer reads are definitely worth more. The question is how much more. Currently you are looking at 50-100x more per base for 454 reads than for Illumina or SOLiD reads.

            On the other hand, people are still doing Sanger sequencing -- that is 50-100X more expensive per base than 454 reads. So it goes back to the fact that there are lots of assays that do not benefit much from vastly more sequence.

            --
            Phillip

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            • #21
              Originally posted by krobison View Post
              The other player in this space will be PacBio; their reads are apparently pushing 5Kb. If you are primarily using long reads to scaffold short ones, then the ~80% single pass accuracy may not be an issue.

              Of course, getting an instrument will set you back a lot more than any of these others . But, there seem to be enough in the field that it is likely one could get access for a project without buying one.
              5kb reads trimmed to 500 usable with QV's in the single digits. There are not entering any real application space for a long time.

              Roche will hold its niche until someone has high quality long reads. It's still a year out or more on the PGM. They need to to something about the cost per base though. Even 454 Jr runs are ridiculous.

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              • #22
                My understanding is that 454 long reads are for whole-genome shotgun only, not amplicon protocol. At least this is the case for the initial release. Which means they won't be useful for 16S diversity studies which is a shame as that would be the biggest win for us (for accurate species level determination).

                Also, as GW_OK pointed out, 454 long reads were announced several years ago and we still don't have a firm shipping date (I heard Q2/Q3 was the latest estimate). Plus you need an instrument upgrade, plus they are not available for 454 Jr (which will annoy a bunch of people). Also it's more like 700bp median than 1kb and the quality drop-off is quite severe towards the read tail.

                454 long reads (i.e. 700 vs 500) may be useful for a limited number of de novo assembly projects but I don't think they are critical, and it is certainly true that the cost is prohibitive for many users.
                Last edited by nickloman; 04-30-2011, 04:34 AM.

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                • #23
                  I heard a rumor that 454 is seriously decreasing its price level in the US. Can anybody confirm this? It is about time too, with the HiSeq making sequencing incredibly cheap...

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                  • #24
                    That would be nice... also would be keen to know if 454 are decreasing their prices.

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                    • #25
                      The problem is that "seriously decrease" could mean reducing the reagent costs by 10%. While welcome, that won't make much of a difference against a >50x price differential.

                      In principle it would not be impossible for Roche to drop the cost/base of 454 sequence to a place where it would be competitive again. At 10-20% of the current cost/base the longer read lengths would make it worthwhile in even large sequencing projects.

                      --
                      Phillip

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                      • #26
                        454 Titanium plus Solexa makes the best assemblies for shotgun metagenomics

                        I have no bias towards 454 Titanium or Solexa, but I have a good amount of experience using them for metagenomic assemblies. Without a doubt, the best assembly emerges from iterations of both technologies (454 first, then solexa seems to work best). I have plenty of contigs in consed to prove it. Where Solexa (75-100 bp) is able to provide great depth and SNP information, it simply will not cover regions of genomes that shotgun 454 titanium can handle. If you look at a typical contig in consed (where I assembled 454 first and solexa after) there can be read depth in the 100'sx and it drops to zero for solexa, only to have 4 or 5 titanium reads connecting this region to the next high depth coverage area. At least a significant part of this is attributable to bad quality scores (sequence trimming removes solexa reads from this area due to crap scores), but even without trimming (i.e. leaving crappy sequences in), these low coverage areas remain.

                        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. 454 Titanium technology clearly has issues with quality, homopolymers, and cost, but it's superior length makes these trade-offs completely worth it. That doesn't mean I'm too loyal to switch to Pac Bio or Ion Torrent if they can compete with the length!

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                        • #27
                          Originally posted by pmiguel View Post
                          The problem is that "seriously decrease" could mean reducing the reagent costs by 10%. While welcome, that won't make much of a difference against a >50x price differential.

                          In principle it would not be impossible for Roche to drop the cost/base of 454 sequence to a place where it would be competitive again. At 10-20% of the current cost/base the longer read lengths would make it worthwhile in even large sequencing projects.
                          Roche's enzyme cascade is inherently more expensive to manufacture and I understand carries some serious royalties to third parties; they probably can come down, but perhaps not enough to make an impact.

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                          • #28
                            Relying on the enzyme cascade could be a thing of the past.

                            http://www.roche.com/media/media_rel...2010-11-01.htm

                            Roche Partners with DNA Electronics to Develop Semiconductor-Based Sequencing System
                            454 Life Sciences, a Roche Company, announced today that it has entered an exclusive partnership with DNA Electronics for the development of a low-cost, high-throughput DNA sequencing system. As part of the agreement, Roche has signed a non-exclusive license for relevant IP from DNA Electronics’ proprietary semiconductor technology portfolio, which enables sensitive detection of nucleotide incorporation during sequencing. The technology will build on 454 Life Sciences’ current pyrosequencing-based sequencing platforms by enabling a seamless evolution from optical detection to inexpensive, highly scalable electrochemical detection. The collaboration leverages 454 Life Sciences’ long read sequencing chemistry with DNA Electronics’ unique knowledge of semiconductor design and expertise in pH-mediated detection of nucleotide insertions, to produce a long read, high density sequencing platform. Financial details of the agreement were not disclosed.

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                            • #29
                              My guess is that DNA Electronics is a very close scheme to Ion Torrent -- so close that Ion Torrent took a license on it just prior to being bought by LIFE. The two questions are how close are they to having a working system & how much of an edge will the existing 454 chemistry give them over Ion.

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                              • #30
                                454 = parallelized licensed Pyrosequencing AB/Biotage IP, Ion Torrent = parallelized licensed DNAe IP. 454 with ISFET = parallelized licensed DNAe IP.

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