DNA Barcodes pp 355-361 | Cite as

Ways to Mix Multiple PCR Amplicons into Single 454 Run for DNA Barcoding

  • Ryuji J. MachidaEmail author
  • Nancy Knowlton
Part of the Methods in Molecular Biology book series (MIMB, volume 858)


Metagenetic analysis using second-generation sequencing offers a novel methodology for measuring the diversity of metazoan communities. Among commercially available second-generation sequencers, the 454 GS FLX Titanium (Roche Diagnostics) offers by far the longest read length and can produce one million sequences from a single run. Compared to the large number of sequences produced from single run, however, number of samples these machines can process is rather low. In this chapter, we describe the use of MID adapters to mix multiple PCR amplicons into a single 454 run. This strategy is rather easy to use and up to 132 samples can be multiplexed into a single 454 run. If a large number of samples are going to be mixed into a single 454 run, however, high cost might be next bottleneck. In this context, we also discuss other ways of multiplexing, including the use of fusion primers and Parallel Tagged Sequencing and weigh their advantages and disadvantages.

Key words

Metagenetics Amplicon sequencing Multiplexing Second-generation sequencer 



We thank David Erickson and W. John Kress for inviting this submission.


  1. 1.
    Venter JC, Remington K, Heidelberg JF et al (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66–74PubMedCrossRefGoogle Scholar
  2. 2.
    DeLong EF, Preston CM, Mincer T et al (2006) Community genomics among stratified microbial assemblages in the ocean’s interior. Science 311:496–503PubMedCrossRefGoogle Scholar
  3. 3.
    Sogin ML, Morrison HG, Huber JA et al (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci USA 103:12115–12120PubMedCrossRefGoogle Scholar
  4. 4.
    Meyer M, Stenzel U, Hofreiter M (2008) Parallel tagged sequencing on the 454 platform. Nat Protoc 3:267–278PubMedCrossRefGoogle Scholar
  5. 5.
    454 Life Sciences (2009) Amplicon fusion primer design guidelines for GS FLX titanium series Lib-A chemistry. 454 Technical Bulletin TCB No.013-2009Google Scholar
  6. 6.
    454 Life Sciences (2010) Rapid library preparation method manual: GS FLX Titanium SeriesGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Invertebrate ZoologySmithsonian Institution, National Museum of Natural HistoryWashingtonUSA

Personalised recommendations