WGA pre-Next Gen Sequencing

Suzanne · 01-21-2009, 04:40 PM

Hi all,
Is anyone using Phi29 amplified genomic DNA for next gen? I am specifically interested in using it before 454 for metagenomic analysis but anyone with experience or opinions with other systems would be helpful.
The newsbot listed a WGA paper but it wasn't analyzed with a next gen instrument so even though it worked great on a bead array, I'd like to know if it has been validated on sequencing platforms.

Thanks,
Suzanne

mgenome · 01-21-2009, 11:57 PM

In the following paper, the authors used the MDA method (phi29 DNA polymerase) and 454 sequencing for viral metagenomics study.
http://biology.plosjournals.org/perl...l.pbio.0040368
PLoS Biology - The Marine Viromes of Four Oceanic Regions

But, MDA may make (maybe significant) quantitative bias on the composition of each member.

For MDA bias, you can referr to a paper reporting selective amplification of ssDNA viruses using MDA.
http://aem.asm.org/cgi/content/abstract/AEM.01275-08v1
In this paper, circular DNA can be amplified very effectively and alter the composition of the amplified DNA.

Suzanne · 01-22-2009, 02:27 PM

Thanks!!
This is a great help.
Suzanne

EPICENTRE · 03-04-2009, 07:24 AM

There was another application by the FBI that looked at various ligation conditions prior to WGA.
http://www.promega.com/geneticidproc...ions/Nunez.pdf

ECO · 03-19-2009, 07:51 AM

Here is another paper for answering this question:

Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.

Suzanne · 03-19-2009, 09:04 PM

Thanks E- This reference is perfect.

S

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01-21-2009, 04:40 PM	#1
Suzanne Registered Vendor Location: Southern California Join Date: Nov 2007 Posts: 10	WGA pre-Next Gen Sequencing Hi all, Is anyone using Phi29 amplified genomic DNA for next gen? I am specifically interested in using it before 454 for metagenomic analysis but anyone with experience or opinions with other systems would be helpful. The newsbot listed a WGA paper but it wasn't analyzed with a next gen instrument so even though it worked great on a bead array, I'd like to know if it has been validated on sequencing platforms. Thanks, Suzanne __________________ MO BIO Laboratories, Inc. Saving You Time for Life

01-21-2009, 11:57 PM	#2
mgenome Junior Member Location: Korea Join Date: Sep 2008 Posts: 5	In the following paper, the authors used the MDA method (phi29 DNA polymerase) and 454 sequencing for viral metagenomics study. http://biology.plosjournals.org/perl...l.pbio.0040368 PLoS Biology - The Marine Viromes of Four Oceanic Regions But, MDA may make (maybe significant) quantitative bias on the composition of each member. For MDA bias, you can referr to a paper reporting selective amplification of ssDNA viruses using MDA. http://aem.asm.org/cgi/content/abstract/AEM.01275-08v1 In this paper, circular DNA can be amplified very effectively and alter the composition of the amplified DNA. Last edited by mgenome; 01-22-2009 at 12:30 AM.

01-22-2009, 02:27 PM	#3
Suzanne Registered Vendor Location: Southern California Join Date: Nov 2007 Posts: 10	Thanks!! This is a great help. Suzanne __________________ MO BIO Laboratories, Inc. Saving You Time for Life

03-19-2009, 09:04 PM	#6
Suzanne Registered Vendor Location: Southern California Join Date: Nov 2007 Posts: 10	Thanks E- This reference is perfect. S __________________ MO BIO Laboratories, Inc. Saving You Time for Life

03-04-2009, 07:24 AM	#4
EPICENTRE Registered Vendor Location: Madison Join Date: Mar 2009 Posts: 5	There was another application by the FBI that looked at various ligation conditions prior to WGA. http://www.promega.com/geneticidproc...ions/Nunez.pdf

03-19-2009, 07:51 AM	#5
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,250	Here is another paper for answering this question: Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.