Barcoded primers used in multiplex amplicon pyrosequencing bias amplification

Appl Environ Microbiol. 2011 Nov;77(21):7846-9. doi: 10.1128/AEM.05220-11. Epub 2011 Sep 2.


"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics*
  • Diagnostic Errors*
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Restriction Fragment Length
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*


  • DNA Primers