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Updated: Jun 24, 2026

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
Methods for generating shotgun and mixed shotgun/paired-end libraries for the 454 DNA sequencer
Graham Wiley1, Simone Macmil, Chunmei Qu
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.
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