Nextera XT for Normalized full-length dscDNA

[lyma223]

Hello everyone,

I am try to make illumine Hiseq 2500 library using normalized full length dscDNA as the start materials. Someone suggested to me using Nextera XT to tagmentation my full length dscDNA. However, I noticed that the enzyme digestion has bias at the ends. My question is that does 50nt near the both ends will not be included or just 3'end?

Thank you very much.

[nucacidhunter]

For transposition, transposase in Nextera needs to interact with DNA which requires some length of DNA and each fragment must be tagmented in both ends for PCR amplification. This results in less coverage for 50 bp from each end. In this case it may not be an issue depending on your sequencing aim and the priming method used for first strand cDNA synthesis (poly T primer or random). Using modified Nextera XT protocol for RNAseq library prep is an established method used with at least Clontech SMARTer Ultra Low Input RNA kit (and Fluidigm C1 Autoprep system) to prepare RNAseq libraries for single-cells. You can get more information from Clontech web site and this link: http://epicentral.blogspot.com.au/20...1_archive.html