SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Using Covaris S2 for RNA shearing shurjo Sample Prep / Library Generation 17 02-03-2016 12:29 PM
Shearing Chromatin using Covaris Bonn Sample Prep / Library Generation 15 08-21-2012 10:28 AM
ChIP - shearing with the Covaris S2 ETHANol Epigenetics 2 08-09-2012 11:47 PM
covaris shearing range chaos81 Illumina/Solexa 1 08-14-2011 05:20 PM
Shearing Amplicons with Covaris lzembek 454 Pyrosequencing 1 01-05-2011 08:34 AM

Reply
 
Thread Tools
Old 12-08-2011, 01:37 AM   #1
babine
Junior Member
 
Location: Gatersleben

Join Date: Dec 2011
Posts: 7
Default Covaris shearing mixture of amplicons

Hi,

I have a project in which I should sequence 16 loci in 96 individuals. Approximately half of the individuals are polyploids so the idea is to use 454 sequencing to avoid cloning. The only thing we didn't really consider is the size of the amplicons ranging between 700 and 2500 bp. A Covaris machine is available and we are planning to use it to shear the amplicons, but the question is: is it feasible to shear amplicons in a mixture (to reduce cost) when the ranging size is so wide?
Does anybody have experience with this kind of setting?

Thank you
babine is offline   Reply With Quote
Old 12-08-2011, 02:05 AM   #2
henry.wood
Member
 
Location: Leeds, UK

Join Date: Apr 2010
Posts: 62
Default

I don't have much experience doing this, but I saw this paper http://www.ncbi.nlm.nih.gov/pubmed/22140562 which seems to address some of your concerns.
henry.wood is offline   Reply With Quote
Old 12-09-2011, 01:08 AM   #3
babine
Junior Member
 
Location: Gatersleben

Join Date: Dec 2011
Posts: 7
Default

Thanks for the paper!

But according to the person responsible for the 454 sequencing in my Institute, there should be no problem for the long fragments, but we might lose a lot of product in the short amplicons (<1 kb). Is that right?
babine is offline   Reply With Quote
Old 12-09-2011, 01:20 AM   #4
henry.wood
Member
 
Location: Leeds, UK

Join Date: Apr 2010
Posts: 62
Default

I can't claim to be much of an expert, I was just passing on a paper that looked relevant. From my understanding the covaris doesn't over-shear too badly, so if you have the right settings, your smaller fragments will hopefully emerge intact. However it's at this stage my knowledge runs out, so I'll have to stand back and let others answer.
henry.wood is offline   Reply With Quote
Old 12-09-2011, 04:38 AM   #5
Heisman
Senior Member
 
Location: St. Louis

Join Date: Dec 2010
Posts: 535
Default

You can test this easily by taking a couple of PCR products at different sizes and fragmenting them individually. Then run them out on a gel and see what the distributions are. If you do one fragment size per sonication it will be clear how they will perform.
Heisman is offline   Reply With Quote
Old 12-09-2011, 05:25 AM   #6
bbeitzel
Member
 
Location: Ft. Detrick, MD

Join Date: Aug 2008
Posts: 50
Default

We have been using Covaris to shear mixed amplicons. There is no problem when the sizes are >~1kb, but smaller fragments require optimization of the Covaris parameters (in general they require a lot more time to be fragmented to the correct size range.) We usually have all amplicons > 1kb or all amplicons <1 kb, so I'm not sure what would happen if you had a mixture of the two sizes and used the small amplicon parameters to shear the mix. I would probably optmize conditions for a small amplicon, then mix in the larger amplicons and see what happens.
bbeitzel is offline   Reply With Quote
Old 12-09-2011, 05:53 AM   #7
babine
Junior Member
 
Location: Gatersleben

Join Date: Dec 2011
Posts: 7
Default

Thank you for your inputs!

We will run a pilot experiment next week with 3 amplicons mixtures: 2 kb, 1 + 0,7 kb and all mixed under different time settings.
We will also consider running a pool of products with size <1kb and an other pool with the rest.

I should have more ideas by next week.
Hopefully it will work...
babine is offline   Reply With Quote
Old 08-24-2012, 01:34 PM   #8
danedwards
Junior Member
 
Location: CT

Join Date: Aug 2012
Posts: 2
Default

I was wondering what progress you made with this? Were you able to optimize for the pooled size groups?
danedwards is offline   Reply With Quote
Reply

Tags
454, amplicon shearing, covaris, size

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 09:45 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO