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Old 04-29-2011, 04:49 PM   #21
SeqAA
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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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Old 04-30-2011, 05:24 AM   #22
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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 at 05:34 AM.
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Old 05-02-2011, 10:47 AM   #23
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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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Old 05-02-2011, 11:48 AM   #24
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That would be nice... also would be keen to know if 454 are decreasing their prices.
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Old 05-02-2011, 01:22 PM   #25
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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.

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Old 05-16-2011, 11:10 AM   #26
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Default 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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Old 05-16-2011, 11:30 AM   #27
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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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Old 05-16-2011, 11:53 AM   #28
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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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Old 05-16-2011, 01:59 PM   #29
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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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Old 05-16-2011, 03:04 PM   #30
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454 = parallelized licensed Pyrosequencing AB/Biotage IP, Ion Torrent = parallelized licensed DNAe IP. 454 with ISFET = parallelized licensed DNAe IP.
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Old 05-17-2011, 02:14 AM   #31
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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

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Old 05-17-2011, 03:00 AM   #32
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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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Old 05-18-2011, 07:30 AM   #33
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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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Old 06-23-2011, 07:53 AM   #34
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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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Old 06-23-2011, 08:26 AM   #35
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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.

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Old 06-23-2011, 02:30 PM   #36
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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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Old 06-23-2011, 05:12 PM   #37
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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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Old 06-24-2011, 07:24 AM   #38
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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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