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Methods for Generating Shotgun and Mixed Shotgun/Paired End ...

Methods for Generating Shotgun and Mixed Shotgun/Paired End ...

Methods for Generating Shotgun and Mixed Shotgun/Paired End

Latest changes dated October 1, 2009Methods for Generating Shotgun and Mixed Shotgun/Paired EndLibraries (with or without MID tags) for the 454 DNA SequencerTitanium Picotiter PlatesGraham Wiley 1 , Simone Macmil 1 , Jing Yi, Ruihua Shi, Chunmei Qu 1 , Yanbo Xing 1 ,Doug White 1 , James D. White 1 , Bruce A. Roe #11Advanced Center for Genome Technology and the Department of Chemistry andBiochemistry, University of Oklahoma, 101 David L. Boren Blvd., Norman, OK 73019# Corresponding AuthorAbstractWith the introduction of massively parallel, microminiature-based instrumentationfor DNA sequencing, robust, reproducible, optimized methods are needed to prepare thetarget DNA for their analysis using these high-throughput approaches because the costper instrument run is orders of magnitude more expensive than for typical Sangerdideoxynucleotide sequencing on fluorescent-based capillary systems. Although themethods provided by 454/Roche with the GS-20 and GS-FLX instruments using theTitanium picotiter plates are robust, in an effort to streamline and automate them, we nowhave incorporated the modifications that we introduced earlier in the FLX protocol intothe most recent Roche Titanium picotiter plate protocol. As a result we have reduced thenumber of manual manipulations, automated portions of the library preparationprocedure, and improved the overall yields for both shotgun and mixed shotgun/pairedend libraries using the chemistry designed for use with the Titanium picotiter plates.Key Words: Pyrosequencing, Shotgun, Paired-End, DNA Library1

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