DNA Barcodes pp 355-361 | Cite as

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

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 858)

Abstract

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 

References

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

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

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