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. Keywords: pyrosequencing; shotgun; paired-end; DNA Library