Unit

UNIT 7.5 High-Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems

  1. Derek Daigle,
  2. Birgitte B. Simen,
  3. Pascale Pochart

Published Online: 1 OCT 2011

DOI: 10.1002/0471142727.mb0705s96

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Daigle, D., Simen, B. B. and Pochart, P. 2011. High-Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems. Current Protocols in Molecular Biology. 96:7.5:7.5.1–7.5.14.

Author Information

  1. 454 Life Sciences, Branford, Connecticut

Publication History

  1. Published Online: 1 OCT 2011
  2. Published Print: OCT 2011

Abstract

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. Curr. Protoc. Mol. Biol. 96:7.5.1-7.5.14. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • high-throughput sequencing;
  • universally tagged libraries;
  • M13 primers;
  • amplicon;
  • multiplexing