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Old 03-19-2014, 04:33 AM   #1
Zwartbie
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Default Ion Chef

Hello,
Has anyone had their Ion Chef installed yet? Do you always have to prepare two chips at a time? I've been reading through the instructions and it seems to rely on you having two Sequencers to run the chips on. It does say chips can be stored for 4.5 hours but a sequencing run is longer than 5 hours so it looks like you can't run the second chip!
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Old 03-19-2014, 09:57 AM   #2
NextGenSeq
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The v3 chemistry only takes a bit over an hour to run.
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Old 03-20-2014, 02:53 AM   #3
MrGuy
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what read length? PE?
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Old 03-20-2014, 07:09 AM   #4
NextGenSeq
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200 SE, the new Ion High-QT will be 400 SE
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Old 03-20-2014, 10:33 AM   #5
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So, let me get this straight: you are saying Ion's new sequencing chemistry will be sequencing SE 200 and 400 bp in 1 hour sequencing runs. Is that correct?
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Old 03-20-2014, 11:09 AM   #6
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Quote:
Originally Posted by MrGuy View Post
So, let me get this straight: you are saying Ion's new sequencing chemistry will be sequencing SE 200 and 400 bp in 1 hour sequencing runs. Is that correct?
No, that is not what he meant. The Ion Chef is Life Techs new sample prep machine for automated library and size selection. The 1 hour is for the library amp step, instead f the 5 hour step now.

The sequencing will still take the 4-5 hours.
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Old 03-20-2014, 11:23 AM   #7
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ok, so it's:
OT2+ES+chip loading --- 5-8 hours
Isothermal+ES+chip loading -- 2 hours
Chef -- 10 hours for two chips
Sequencing still at 4 hours

In other words, with Chef, 18-20 hours would produce two 200 or 400 bp SE runs on one sequencer or ~15 hours for two sequencers

Or with IsoChef (if that is for real) then as little as 6 hours for two runs with two machines. That's a regular work shift.

Extrapolate on that...
DNA extraction - 30 min
Exome Ampliseq 2 hrs??
Lunch 2 hours :-)
IsoChef -- 2 hours
200 bp run -- 3hrs??
Autoanalysis on torrent server

Exome in a day. That's crazy. Wonder if they will really ever get there.
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Old 03-20-2014, 11:35 AM   #8
skbrimer
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Close, I think you just have your terms jumbled.

workflow now:
extract-->OT2+ES+Chipload-->PGM/ION --->
30min --> 5-7hours --> 4ish hrs extraction and sequecning one chip in a day

Workflow early adaptors: still one chip in a day
extract --> Isolthermal+ES+chip loading ---> PGM/ION
30min ---> 2hours ----> 4ish hrs

Workflow with Chef:two chips in a day
extract ---> IonChef ---> PGM
30-60 min 2hrs 4ish hrs

The chef is suppose to be the isothermal+ES+Chipload all in one hands free device.
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Old 07-21-2014, 11:06 AM   #9
madebeljak
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Hi,

Has anyone tested out Ion Chef? If so, what are your thoughts on it?
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Old 07-22-2014, 07:46 AM   #10
NextGenSeq
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We get improved loading and more uniform results.

Our biggest issue is consistent quantification of libraries for emulsion PCR.
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Old 07-22-2014, 12:48 PM   #11
madebeljak
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What do you use to quantify your library for emulsion PCR?
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Old 07-29-2014, 09:17 AM   #12
NextGenSeq
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We use the Nanodrop and/or the Bioanalyzer to normalize to a library which gave good sequencing results.

This works for the most part. We used to use QPCR but the adaptors we are using don't work with the LifeTech QPCR probe.
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Old 07-29-2014, 09:26 AM   #13
madebeljak
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In case you are looking to try something else, we use Qubit fluorometer and dsDNA High Sensitivity reagents. It is really quick assay and our experience has been great! I would definitely recommend over the Nanodrop.
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Old 08-07-2014, 01:08 AM   #14
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Quote:
Originally Posted by madebeljak View Post
In case you are looking to try something else, we use Qubit fluorometer and dsDNA High Sensitivity reagents. It is really quick assay and our experience has been great! I would definitely recommend over the Nanodrop.
Same here, Nanodrop can be off by 50%, Qubit never is.
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