Methods for Generating Shotgun and Mixed Shotgun/Paired End Libraries (with or without MID tags) for the 454 DNA Sequencer Titanium Picotiter Plates

With the introduction of massively parallel, microminiature-based instrumentation for DNA sequencing, robust, reproducible, optimized methods are needed to prepare the target DNA for their analysis using these high-throughput approaches because the cost per instrument run is orders of magnitude more expensive than for typical Sanger dideoxynucleotide sequencing on fluorescent-based capillary systems. Although the methods provided by 454/Roche with the GS-20 and GS-FLX instruments using the Titanium picotiter plates are robust, in an effort to streamline and automate them, we now have incorporated the modifications that we introduced earlier in the FLX protocol into the most recent Roche Titanium picotiter plate protocol. As a result we have reduced the number of manual manipulations, automated portions of the library preparation procedure, and improved the overall yields for both shotgun and mixed shotgun/paired end libraries using the chemistry designed for use with the Titanium picotiter plates.