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Methods for Generating Shotgun and Mixed Shotgun/Paired‐End Libraries for the 454 DNA Sequencer

With the introduction of massively parallel, microminiature‐based instrumentation for DNA sequencing, robust, reproducible, optimized methods are needed to prepare the target DNA for analysis using these high‐throughput approaches because the cost per instrument run is orders of magnitude more than for typical Sanger dideoxynucleotide sequencing on fluorescence‐based capillary systems. The methods provided by the manufacturer for genome sequencing using the 454/Roche GS‐20 and GS‐FLX instruments are robust. However, in an effort to streamline them for automation, we have incorporated several novel changes and deleted several extraneous steps. As a result of modifying these sample preparation protocols, the number of manual manipulations has also been minimized, and the overall yields have been improved for both shotgun and mixed shotgun/paired‐end libraries. Curr. Protoc. Hum. Genet. 61:18.1.1‐18.1.21. © 2009 by John Wiley & Sons, Inc.