[pmiguel]

I guess increasing read length can only go so far as there's a limit to what length insert can be successfully brPCR'd.

Not sure this is a major issue. We did a MiSeq run with >1 kb insert amplicons. Actually the average insert sizes looked more like 1.5 kb, or even larger, but after mapping the PE reads back to an earlier de novo assembly from the same organism, the size came back just over 1 kb for most of them.

We did cluster at a pretty low density, though. See this thread for details.

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Phillip

[MrGuy]

Internally, Illumina have run 400bp with the same 250bp chemistry and instrument upgrades and hit 80% >Q30 at 400bp (which I think is totally acceptable when comparing to 454 data - especially considering read overlap increasing the score further).

I'm not overly worried about the chemistry, more so the box and software. The issue is pretty well defined:

http://seqanswers.com/forums/showthread.php?t=17465

Finding a workaround to make a tool work means that I have chosen the wrong tool.

[krobison]

I'm not overly worried about the chemistry, more so the box and software. The issue is pretty well defined:

http://seqanswers.com/forums/showthread.php?t=17465

Finding a workaround to make a tool work means that I have chosen the wrong tool.

If you are completely fighting the tool, then I can agree. But with the MiSeq upgrade to image both surfaces, you have a lot of capacity to burn on spiked-in phiX, and putting an internal standard in is hardly unusual or a workaround.

[bbeitzel]

Finding a workaround to make a tool work means that I have chosen the wrong tool.

Or the right tool doesn't exist yet, so you make do with what you have...

[ECO]

Confirming a couple of things from my upgrade documents...most notably from my standpoint is that the official new tile number is 14 for each surface...so 28 total up from the previous 12.

[jrbell71]

Updates to TSCA are coming at the end of the month in DesignStudio:

-Increased plexity up to 1,536 amplicons per reaction – designs covering up to ~600 kb of total content

-More flexible amplicon sizes – smaller to enable custom panel support for FFPE, and larger to take advantage of 2 × 250 bp read lengths supported on the MiSeq system

-Designs for non-human genomes – Early access to selectable reference genomes for mouse, rat, and bovine

[csquared]

I'm not overly worried about the chemistry, more so the box and software. The issue is pretty well defined:

http://seqanswers.com/forums/showthread.php?t=17465

Finding a workaround to make a tool work means that I have chosen the wrong tool.

With the MiSeq software update, you can use a matrix from a different run, supposedly allowing low diversity/amplicon sequencing on the platform. Time will tell how effective that solution will be, but should work.....in theory.....

No lost reads to PhiX and no workaround. Should help with inline barcodes and other such things.

[krobison]

How do the prices of the 2x250 kits compare to the prior prices for 2x150 kits?

[pmiguel]

v2 kits list for $700, $900, $1000 for 50, 150 and 250 cycle kits, respectively. Well, $5 less that that, but you get the idea.

--
Phillip

[GenoMax]

Our MiSeq was upgraded yesterday. So there are "n+1" machines out there now that physically have the new hardware capable of 2 x 250 bp runs.

Hopefully will have something to report on an actual run soon.

[TonyBrooks]

With the MiSeq software update, you can use a matrix from a different run, supposedly allowing low diversity/amplicon sequencing on the platform. Time will tell how effective that solution will be, but should work.....in theory.....

No lost reads to PhiX and no workaround. Should help with inline barcodes and other such things.

See this blog post by Nick Loman. I guess they have implemented the settings described directly into MiSeq Control Software. There should still be some (5%) spike in to fix other low diversity issues though, no?

[ramsemm]

With the MiSeq software update, you can use a matrix from a different run, supposedly allowing low diversity/amplicon sequencing on the platform. Time will tell how effective that solution will be, but should work.....in theory.....

No lost reads to PhiX and no workaround. Should help with inline barcodes and other such things.

Do you know if this ability to use a matrix from a different run came with the most recent MCS upgrade to MCS 2.0.0.1? If so, can it be used on a machine that hasn't yet received the hardware upgrade?

Thanks!
Molli

[behavin]

Do you know if this ability to use a matrix from a different run came with the most recent MCS upgrade to MCS 2.0.0.1? If so, can it be used on a machine that hasn't yet received the hardware upgrade?

Thanks!
Molli

According to our FAS, as long as the software is updated you are able to set the "good" matrix, as well as run the 2x250. You won't get the increased data outputs and faster cycle times until the hardware upgrade however.

[james hadfield]

Our upgrade is booked in for next week. looking forward to 20M reads!

[NGSsequencer]

Does anyone have any public data sets available from their upgraded Miseq?
I'd love to see the quality metrics.