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.
University of Oklahoma, Norman, Oklahoma, USA.
Graham
G
Wiley
Simone
S
Macmil
Chunmei
C
Qu
Ping
P
Wang
Yanbo
Y
Xing
Doug
D
White
Jianfeng
J
Li
James D
JD
White
Alexander
A
Domingo
Bruce A
BA
Roe
D004247:DNA / Q000737:chemistry; D015723:Gene Library; D023281:Genomics / Q000379:methods; D008957:Models, Genetic; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods
D004247:DNA