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Accurate multiplexing and filtering for high-throughput amplicon-sequencing.
Journal article

Accurate multiplexing and filtering for high-throughput amplicon-sequencing.

  • Esling P Department of Genetics and Evolution, University of Geneva, Sciences 3, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland IRCAM, UMR 9912, Université Pierre et Marie Curie, Paris, France philippe.esling@unige.ch.
  • Lejzerowicz F Department of Genetics and Evolution, University of Geneva, Sciences 3, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
  • Pawlowski J Department of Genetics and Evolution, University of Geneva, Sciences 3, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
  • 2015-02-19
Published in:
  • Nucleic acids research. - 2015
Algorithms
Computer Simulation
DNA Primers
Gene Library
High-Throughput Nucleotide Sequencing
Internet
Multiplex Polymerase Chain Reaction
Reproducibility of Results
Software
English 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.
Language
  • English
Open access status
gold
Identifiers
Persistent URL
https://sonar.ch/global/documents/254952
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