Accurate multiplexing and filtering for high-throughput amplicon-sequencing

Nucleic Acids Res. 2015 Mar 11;43(5):2513-24. doi: 10.1093/nar/gkv107. Epub 2015 Feb 17.


Tagging amplicons with tag sequences appended to PCR primers allow the multiplexing of numerous samples for high-throughput sequencing (HTS). This approach is routinely used in HTS-based diversity analyses, especially in microbial ecology and biomedical diagnostics. However, amplicon library preparation is subject to pervasive sample sequence cross-contaminations as a result of tag switching events referred to as mistagging. Here, we sequenced seven amplicon libraries prepared using various multiplexing designs in order to measure the magnitude of this phenomenon and its impact on diversity analyses. Up to 28.2% of the unique sequences correspond to undetectable (critical) mistags in single- or saturated double-tagging libraries. We show the advantage of multiplexing samples following Latin Square Designs in order to optimize the detection of mistags and maximize the information on their distribution across samples. We use this information in designs incorporating PCR replicates to filter the critical mistags and to recover the exact composition of mock community samples. Being parameter-free and data-driven, our approach can provide more accurate and reproducible HTS data sets, improving the reliability of their interpretations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Computer Simulation
  • DNA Primers / genetics*
  • Gene Library*
  • High-Throughput Nucleotide Sequencing / methods*
  • Internet
  • Multiplex Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Software


  • DNA Primers