Possibility of tagmentation without fragmentation with Nextera XT kit

Ahmed_BH · 07-11-2014, 06:28 AM

Hello everyone,

I would like to know if it's possible to do the fragmentation of my cDNA amplicon without any fragmentation using the Nextera XT kit. PCR amplicons have already sizes between 150 and 600 bp. these PCR products will be sequenced with Miseq.

Thanks in advance

Olaf Blue · 07-11-2014, 06:45 AM

Hello Ahmed_BH,

Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base scission/cut at the site, which after the PCR to add adapters, generates a 9-base duplication at each insertion site. but the scission/fragmentation is a primary characteristic of Transposome insertion.

Ahmed_BH · 07-11-2014, 06:58 AM

Quote:

Originally Posted by Olaf Blue

Hello Ahmed_BH,

Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base scission/cut at the site, which after the PCR to add adapters, generates a 9-base duplication at each insertion site. but the scission/fragmentation is a primary characteristic of Transposome insertion.

thanks for answers
so you think that it's possible to use any kit which use fragmentation/tagmentation step.
the particularity of my PCR products that i have ligated a specific index for everyone sample and then i have pooled all the samples. i want now to keep these index during the preparation of libraries for Miseq sequencing.

Olaf Blue · 07-11-2014, 07:11 AM

The Nextera technology is unique. If you have PCR products that have ligated adapters/indexes, a tagmentation reaction will fragment the products and depending on the length of the PCR products, may well lose one of the adapter ends, which will be replaced by an adapter from the Nextera process.

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07-11-2014, 06:28 AM	#1
Ahmed_BH Junior Member Location: Tunisia Join Date: Jul 2014 Posts: 4	Possibility of tagmentation without fragmentation with Nextera XT kit Hello everyone, I would like to know if it's possible to do the fragmentation of my cDNA amplicon without any fragmentation using the Nextera XT kit. PCR amplicons have already sizes between 150 and 600 bp. these PCR products will be sequenced with Miseq. Thanks in advance

07-11-2014, 06:45 AM	#2
Olaf Blue Member Location: Madison, WI Join Date: Nov 2010 Posts: 58	Hello Ahmed_BH, Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base scission/cut at the site, which after the PCR to add adapters, generates a 9-base duplication at each insertion site. but the scission/fragmentation is a primary characteristic of Transposome insertion.

07-11-2014, 07:11 AM	#4
Olaf Blue Member Location: Madison, WI Join Date: Nov 2010 Posts: 58	The Nextera technology is unique. If you have PCR products that have ligated adapters/indexes, a tagmentation reaction will fragment the products and depending on the length of the PCR products, may well lose one of the adapter ends, which will be replaced by an adapter from the Nextera process.