[flxlex]

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...

[nickloman]

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.

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.

[pmiguel]

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

[GW_OK]

Your reads were over 100kb, very accurate and you had a mountain of them ?

Are you an agent of a Nanopore company?

[clivey]

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).

[kmcarr]

Then I'd be dreaming

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?

[clivey]

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

[clivey]

What if

Your reads were over 100kb, very accurate and you had a mountain of them ?

[GW_OK]

It's taken Roche way to long to launch this and they are too quiet regarding any other plans they may have. I believe they have licensed the same semiconductor tech as Ion Torrent so I'm waiting for an announcement one day of a similar platform....

I absolutely agree. I remember them first talking about 1k reads way back in 2007-8 or whenever Titanium was first coming out. It was always going to drop in the next 6 months and was then always pushed back.
And we were always, always asking about when they were going to automate emPCR. I wonder if they can license the OneTouch from iotorrent, lol.
The 454 is quickly becoming the has-been of the field.

[krobison]

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.

[niceday]

it would also be useful if roche automated their emulsion pcr-breaking and enrichment steps. SOLiD have the ez system, ion torrent will have the one touch. Doing any of these processes manually is a right pain. I'm suprised roche haven't done something about this.

[petang]

How about PacBio machine that claimed a maximun read length of 3kb with an average of 1kb? The only problem is they can only generate ~80000 reads on a 30 min run.

[Michael.James.Clark]

Plant transcriptomics. With so many recent segmental or whole genome duplications in plants assembling paralogous transcripts from shorter reads can be maddening.

How much will 1kb reads help with that versus 400bp reads?

There are probably better solutions than longer reads for such situations. For example, unique tagging every transcript in individual droplets during library generation.

[RCJK]

I've been told the Titanium XL+ upgrade should be launched by the end of May, but don't have any more details than that other than that new computing is required to process the data.
It's taken Roche way to long to launch this and they are too quiet regarding any other plans they may have. I believe they have licensed the same semiconductor tech as Ion Torrent so I'm waiting for an announcement one day of a similar platform....