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High‐Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems

2011; Wiley; Volume: 96; Issue: 1 Linguagem: Inglês

10.1002/0471142727.mb0705s96

1934-3639

Derek A. Daigle, Birgitte B. Simen, Pascale Pochart,

Bacteriophages and microbial interactions

This unit describes a method to convert PCR products (amplicons) flanked by universal M13 primers into a library for use on all 454 Sequencing Systems (454 Life Sciences, a Roche Company). This is especially useful for simultaneous sequencing and analysis of large numbers of amplicons or for the detection of minor variations within the amplified products. The method described here involves preparing a library of DNA with specific primers containing adaptor sequences recognized by the GS Junior System sequencing process. The data from the sequencing run are processed and analyzed by 454 Life Sciences software. This approach allows for multiplexing of a large number of amplicons to streamline processing and analysis. Any pre-existing universally tagged amplicon, primer set, or plasmid with M13 sequences flanking the cloning site can be used in this protocol.