nextera

niceday · 09-03-2010, 06:08 AM

Has anyone used Nextera library prep for illumina? I'm looking to test but any info on biases or other data would be welcome.

NextGenSeq · 09-03-2010, 07:29 AM

We tried it. If you don't have a Covaris it might be worthwhile. It is very easy and fast but it doesn't give a tight size distribution.

niceday · 09-03-2010, 07:32 AM

Thanks.

We do have a covaris but it's the price. We have to charge a wet lab hourly rate. If nextera can cut 2-4 hours off library making it might be useful.
I suppose we could use an ez-gel to tighten the size range.

epibio · 09-09-2010, 12:55 PM

You can control the size distribution of the library, based on the buffer used (see our blog post for Bioanalyzer traces). If you want to tighten up the size range further, you could use Ampure XP.

epistatic · 09-09-2010, 01:14 PM

I would use the HMW buffer, never the LMW buffer for Illumina prep. The peak at 200 represents 135 bp of the Nextera adapters and 65 bp of insert. I have had to use the HMW buffer and do size selection. Be careful if you do Size Selection post PCR, you do not have overages to re-amplify and the standard PE PCR Primers will not work.

epibio · 09-10-2010, 11:31 AM

Starting with the recommended amount of DNA for Nextera (50 ng), you should get 300-500 ng of "tagmented" DNA after 9 cycles of PCR. We have some customers who don't bother with size selection, and get good sequencing results--they find that a tight size distribution is not necessary for Illumina sequencing in their application. However, it's your choice.

Also, in response to the original point, Nextera will definitely save 2-4 hours compared to Covaris + adaptor ligation.

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09-03-2010, 06:08 AM	#1
niceday Member Location: cambridge Join Date: Apr 2010 Posts: 68	nextera Has anyone used Nextera library prep for illumina? I'm looking to test but any info on biases or other data would be welcome.

09-03-2010, 07:29 AM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	We tried it. If you don't have a Covaris it might be worthwhile. It is very easy and fast but it doesn't give a tight size distribution.

09-03-2010, 07:32 AM	#3
niceday Member Location: cambridge Join Date: Apr 2010 Posts: 68	Thanks. We do have a covaris but it's the price. We have to charge a wet lab hourly rate. If nextera can cut 2-4 hours off library making it might be useful. I suppose we could use an ez-gel to tighten the size range.

09-09-2010, 12:55 PM	#4
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	You can control the size distribution of the library, based on the buffer used (see our blog post for Bioanalyzer traces). If you want to tighten up the size range further, you could use Ampure XP.

09-09-2010, 01:14 PM	#5
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	I would use the HMW buffer, never the LMW buffer for Illumina prep. The peak at 200 represents 135 bp of the Nextera adapters and 65 bp of insert. I have had to use the HMW buffer and do size selection. Be careful if you do Size Selection post PCR, you do not have overages to re-amplify and the standard PE PCR Primers will not work.

09-10-2010, 11:31 AM	#6
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	Starting with the recommended amount of DNA for Nextera (50 ng), you should get 300-500 ng of "tagmented" DNA after 9 cycles of PCR. We have some customers who don't bother with size selection, and get good sequencing results--they find that a tight size distribution is not necessary for Illumina sequencing in their application. However, it's your choice. Also, in response to the original point, Nextera will definitely save 2-4 hours compared to Covaris + adaptor ligation.