life sciences prep challenge -- thoughts?

delinquentme · 12-03-2011, 07:08 PM

So I've been checking out the life sciences grand challenge : speed

( specifically this challenge is to reduce the prep time involved in the DNA of these machines from 10 to 4 hours )

I'm looking at you guys as some of the best in the business as I don't have access to these machines.

It seems that the most significant areas which could contribute to time cuts would be the PCR ( using preheated baths / microfluidics ) and prepping the input sample as to give the correct DNA concentration in the yields.

I'm interested in what the folks around here have to say about how difficult this would be or if you'd happen to have recommendations as to what protocol to hack for the best returns on time reduction.

epistatic · 12-09-2011, 09:07 AM

For libraries that I generate, PCR is the quickest and easiest step. There is no real reason to overly complicate this step with microfluidics. A standard tcycler is cheap and found in every lab already.

Most NGS DNA libraries:

DNA -> fragment, clean up -> multiple enzyme steps with clean ups (End repair, dA tail, ligation, etc) -> size selection (gel, Pippin prep) -> quick low cycle PCR -> library validation with BioA, qPCR.

The best and quickest method has been what Epicentre came up with, Nextera. DNA -> fragmentation to ligation in one quick and easy step -> quick low cycle PCR -> validation.

It will be hard to get better and easier than that...

delinquentme · 12-09-2011, 09:44 AM

so lets break this down into steps

1) Nextera. DNA
This kit ? It mentions Illumina .. does it work for the Ion PGM?

2) Fragmentation to ligation in one quick and easy step
This sounds like its part of the kit? or a machine operation?

3) quick low cycle PCR
Did some googling and this doesn't seem like its run-of-the-mill PCR ... So do I need a special machine?

4) validation
I think this is the quantitation step ( the DNA / liquid ratio )?

BTW ! thanks for digging back into these articles!

NextGenSeq · 12-09-2011, 10:10 AM

The Nextera protocol gives very broad size ranges. The first time we ran it the library looked so strange we didn't even sequence it.

delinquentme · 12-09-2011, 10:16 AM

yeahh I think the PGM needs really specific lengths too?

epistatic · 12-09-2011, 11:57 AM

Nextera was developed for 454, Illumina, et al. It could be used for the PGM, you do the library prep and do a size selection.

For your question, you mention PCR. Do you mean PCR or the emulsion PCR?

delinquentme · 12-09-2011, 12:34 PM

so im lost right now is low-cycle PCR == emulsion PCR?

Im 90% sure that the sequencer needs consensus sequences so it requires amplification .. but I was hoping I could get some specific PGM knowledge here with regards to what is needed

epistatic · 12-16-2011, 12:29 PM

PCR is needed in the library prep and emPCR is needed to put the library onto the beads for sequencing.

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12-03-2011, 07:08 PM	#1
delinquentme Member Location: pittsburgh Join Date: Jul 2011 Posts: 12	life sciences prep challenge -- thoughts? So I've been checking out the life sciences grand challenge : speed ( specifically this challenge is to reduce the prep time involved in the DNA of these machines from 10 to 4 hours ) I'm looking at you guys as some of the best in the business as I don't have access to these machines. It seems that the most significant areas which could contribute to time cuts would be the PCR ( using preheated baths / microfluidics ) and prepping the input sample as to give the correct DNA concentration in the yields. I'm interested in what the folks around here have to say about how difficult this would be or if you'd happen to have recommendations as to what protocol to hack for the best returns on time reduction.

12-09-2011, 09:44 AM	#3
delinquentme Member Location: pittsburgh Join Date: Jul 2011 Posts: 12	so lets break this down into steps 1) Nextera. DNA This kit ? It mentions Illumina .. does it work for the Ion PGM? 2) Fragmentation to ligation in one quick and easy step This sounds like its part of the kit? or a machine operation? 3) quick low cycle PCR Did some googling and this doesn't seem like its run-of-the-mill PCR ... So do I need a special machine? 4) validation I think this is the quantitation step ( the DNA / liquid ratio )? BTW ! thanks for digging back into these articles! Last edited by delinquentme; 12-09-2011 at 09:46 AM. Reason: added thanks!

12-09-2011, 09:07 AM	#2
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	For libraries that I generate, PCR is the quickest and easiest step. There is no real reason to overly complicate this step with microfluidics. A standard tcycler is cheap and found in every lab already. Most NGS DNA libraries: DNA -> fragment, clean up -> multiple enzyme steps with clean ups (End repair, dA tail, ligation, etc) -> size selection (gel, Pippin prep) -> quick low cycle PCR -> library validation with BioA, qPCR. The best and quickest method has been what Epicentre came up with, Nextera. DNA -> fragmentation to ligation in one quick and easy step -> quick low cycle PCR -> validation. It will be hard to get better and easier than that...

12-09-2011, 10:10 AM	#4
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	The Nextera protocol gives very broad size ranges. The first time we ran it the library looked so strange we didn't even sequence it.

12-09-2011, 10:16 AM	#5
delinquentme Member Location: pittsburgh Join Date: Jul 2011 Posts: 12	yeahh I think the PGM needs really specific lengths too?

12-09-2011, 11:57 AM	#6
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	Nextera was developed for 454, Illumina, et al. It could be used for the PGM, you do the library prep and do a size selection. For your question, you mention PCR. Do you mean PCR or the emulsion PCR?

12-09-2011, 12:34 PM	#7
delinquentme Member Location: pittsburgh Join Date: Jul 2011 Posts: 12	so im lost right now is low-cycle PCR == emulsion PCR? Im 90% sure that the sequencer needs consensus sequences so it requires amplification .. but I was hoping I could get some specific PGM knowledge here with regards to what is needed

12-16-2011, 12:29 PM	#8
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	PCR is needed in the library prep and emPCR is needed to put the library onto the beads for sequencing.