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Curr Protoc Hum Genet. 2009 Apr;Chapter 18:Unit18.1. doi: 10.1002/0471142905.hg1801s61.

Methods for generating shotgun and mixed shotgun/paired-end libraries for the 454 DNA sequencer.

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  • 1University of Oklahoma, Norman, Oklahoma, USA.

Abstract

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.

(c) 2009 by John Wiley & Sons, Inc.

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