Fearing that 454 failed to compete.

[rskr]

454 seems to have failed to compete. While the short read technology was developing, there were certain tradeoffs between longer reads with errors, for many common applications, so indeed there was a niche for 454, however these days short reads aren't so short, but 454 never seemed correct there errors. , and even for assemblies where length is an obvious advantage they still seem to depend on their competition for correction, whereas sipe and lipe have seemed to obviate the need for longer reads. Is 454 actively developing new chemistry that is going to be competitive, or are their machines going to wither on the vine?

[rskr]

No takers?

It is sad, Illumina really needs the competition.

[pmiguel]

Well, there is still lots of stuff being done on 3730's that probably should migrate to 454s. Although the amount of sequence generated per run by a 454 is trivial compared to an Illumina, it looks great compared to a 3730XL. A single 454 run generates as much sequence as a year's worth of 3730XL runs.

Sure there are some assays that still need the Sanger touch, but much of the reason people are still running those old Sanger war horses are that they don't want to have to deal with migrating their methodology from 3730XL to 454.

Will that save the 454? I think the next 6 months or so will tell. Their GS-FLX+ tech should be completely rolled out by then. If it looks good, that will probably encourage the Sanger-ites to convert and Roche could have another couple of years before Ion Torrent, Pac Bio, or some other technology completely eats their lunch. If, on the other hand, GS-FLX+ ends up having big problems, then yeah, I would say it is over for the 454.

--
Phillip

[rskr]

Will that save the 454? I think the next 6 months or so will tell. Their GS-FLX+ tech should be completely rolled out by then. If it looks good, that will probably encourage the Sanger-ites to convert and Roche could have another couple of years before Ion Torrent, Pac Bio, or some other technology completely eats their lunch. If, on the other hand, GS-FLX+ ends up having big problems, then yeah, I would say it is over for the 454.

--
Phillip

http://454.com/products/gs-flx-system/index.asp

I guess they are addressing the issue of the complicated error model by depth of coverage? What about ploidy?

[ajthomas]

In spite of the short read technologies getting longer, their read lengths still can't compare to that achieved on the 454. Of course, those longer "short" reads also means the number of applications where the 454 is required is shrinking. In my own case, I must have at least 400bp reads, and I'm excited about the longer reads of the FLX+ because that opens up the way for some other experiments I couldn't do before. It will be a while (maybe a long while) before the short read technologies can do what I need.

[rskr]

In spite of the short read technologies getting longer, their read lengths still can't compare to that achieved on the 454. Of course, those longer "short" reads also means the number of applications where the 454 is required is shrinking. In my own case, I must have at least 400bp reads, and I'm excited about the longer reads of the FLX+ because that opens up the way for some other experiments I couldn't do before. It will be a while (maybe a long while) before the short read technologies can do what I need.

Oh do tell.

I find 100 base paired end data do subsume just about any benefits gained from error prone reads with a mean of 400 but a median of 200.

[ajthomas]

I'm using it primarily for genotyping highly polymorphic genes (MHC if you must know), sequencing amplicons of 200-400bp long. Because some alleles only differ by one or two bases that may be at one end or the other of the amplicon, reads that are not full length cannot always differentiate some closely-related alleles. I must have full-length reads of my amplicons which I cannot get from any NGS technology except 454.

[rskr]

I'm using it primarily for genotyping highly polymorphic genes (MHC if you must know), sequencing amplicons of 200-400bp long. Because some alleles only differ by one or two bases that may be at one end or the other of the amplicon, reads that are not full length cannot always differentiate some closely-related alleles. I must have full-length reads of my amplicons which I cannot get from any NGS technology except 454.

And you can do that with the 454 error model? Last I checked a mean quality of 30 would guarantee several errors in a 200-400bp read? Might be better off With a 250 base insert size and 150 bp paired end reads, with an overlapper that finds the intersection.

[ajthomas]

It works just fine. Perhaps the accuracy is better than you think. I don't see nearly as many errors as you imply here. And no, I can't work with shorter reads that must be overlapped. I had to do that before switching from the older standard chemistry to the Titanium chemistry. I ended up with a number of allele misidentifications because of it. Some of the alleles are just too similar and can't be reliably identified without a full-length sequence. Say you have four different alleles: A and B differ from C and D by one base near the 5' end. A and C differ from B and D by one base near the 3' end. Any given sample may have any combination of the four (there are ~10 loci in the genome, so ~20 alleles present in a heterozygote). You can't differentiate these four alleles without having both ends of the amplicon on the same read.

[rskr]

without having both ends of the amplicon on the same read.

'''''what paired end is

[ajthomas]

I'm not trying to argue with you, but I've looked at the options and the 454 is the only one that works for my application. I can't get 400bp reads any other way, and one of my amplicons is nearly that long.

[rskr]

I'm not trying to argue with you, but I've looked at the options and the 454 is the only one that works for my application. I can't get 400bp reads any other way, and one of my amplicons is nearly that long.

Certainly you have some will to analyze erroneous data. If you cared you would have seen that 454 is only .9999% accurate with deep coverage, but you are talking about analyzing individual reads looking for variants, which suggests a different type of of "work for you", than I would find acceptable, but hey you are probably an MD analyzing a major histocompatibility complex, so you can get away with saying anything you want, because you hate statistics.

[ajthomas]

Does the fact that I use the 454 offend you or something? I explained why I use it and why other technologies aren't appropriate for my work and you seem to think I'm an idiot for using it. I'm a little confused at your derision.

By the way, I don't look at individual reads, I look at consensus reads (usually 10-100X coverage per variant).

[ECO]

Certainly you have some will to analyze erroneous data. If you cared you would have seen that 454 is only .9999% accurate with deep coverage, but you are talking about analyzing individual reads looking for variants, which suggests a different type of of "work for you", than I would find acceptable, but hey you are probably an MD analyzing a major histocompatibility complex, so you can get away with saying anything you want, because you hate statistics.

Arguments + facts + opinions please. Save the insults. Thanks.

[rskr]

Does the fact that I use the 454 offend you or something? I explained why I use it and why other technologies aren't appropriate for my work and you seem to think I'm an idiot for using it. I'm a little confused at your derision.

By the way, I don't look at individual reads, I look at consensus reads (usually 10-100X coverage per variant).

So, what is the difference between that and looking at a pileup of paired end Illumina reads? They will get the linkage just as well.