SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Ion Torrent



Similar Threads
Thread Thread Starter Forum Replies Last Post
Cloud Computing for the Life Sciences mza Events / Conferences 6 10-24-2012 01:32 AM
Life Grand Challenge crowd sourcing model - a critique lek2k Ion Torrent 0 08-18-2011 05:49 AM
Spanning the Spectrum of Life Sciences Analytics (March 18, 2010) JMP Genomics Events / Conferences 1 03-08-2010 02:05 PM
454 Life Sciences Celebrates a Milestone in Genomic Sequencing james hadfield General 0 07-31-2009 01:53 AM

Reply
 
Thread Tools
Old 12-03-2011, 07:08 PM   #1
delinquentme
Member
 
Location: pittsburgh

Join Date: Jul 2011
Posts: 12
Default 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.
delinquentme is offline   Reply With Quote
Old 12-09-2011, 09:07 AM   #2
epistatic
Senior Member
 
Location: Dronning Maud Land

Join Date: Mar 2009
Posts: 129
Default

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...
epistatic is offline   Reply With Quote
Old 12-09-2011, 09:44 AM   #3
delinquentme
Member
 
Location: pittsburgh

Join Date: Jul 2011
Posts: 12
Default

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!
delinquentme is offline   Reply With Quote
Old 12-09-2011, 10:10 AM   #4
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 482
Default

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.
NextGenSeq is offline   Reply With Quote
Old 12-09-2011, 10:16 AM   #5
delinquentme
Member
 
Location: pittsburgh

Join Date: Jul 2011
Posts: 12
Default

yeahh I think the PGM needs really specific lengths too?
delinquentme is offline   Reply With Quote
Old 12-09-2011, 11:57 AM   #6
epistatic
Senior Member
 
Location: Dronning Maud Land

Join Date: Mar 2009
Posts: 129
Default

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?
epistatic is offline   Reply With Quote
Old 12-09-2011, 12:34 PM   #7
delinquentme
Member
 
Location: pittsburgh

Join Date: Jul 2011
Posts: 12
Default

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
delinquentme is offline   Reply With Quote
Old 12-16-2011, 12:29 PM   #8
epistatic
Senior Member
 
Location: Dronning Maud Land

Join Date: Mar 2009
Posts: 129
Default

PCR is needed in the library prep and emPCR is needed to put the library onto the beads for sequencing.
epistatic is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 05:24 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO