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  • Improved protocol for 454 Titanium using SMARTer RACE

    Hi everyone.
    I am working with some transciptome data that were obtained using an improved protocol for the 454 titanium technology (http://www.bio.utexas.edu/research/m...%2012-18-9.pdf).

    Under this protocol we have four different pools of cDNAs that bear different set of adaptors and I am struggling to figure out the adaptors that I have to remove from the raw data. According to the Figure 1 of the protocol, the primer Btitn+halfswitch only should be presented in the 5' end (plus strand); however, I can find that adaptor in both strands and I dont understand why!.

    I have been using CLC to import the sff file and use the clipping information. After this, the 5' end seems to be clean. However, when I look for the different adaptors to be sure that everything has been removed I still find them. In addition, I have overrepresented sequences that include the Cap-primer.

    Does somebody use that protocol before?.
    Any feedback is more than welcome.

    Thanks!
    p.

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