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Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

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  • Ulrike Menzel
  • Victor Greiff
  • Tarik A Khan
  • Ulrike Haessler
  • Ina Hellmann
  • Simon Friedensohn
  • Skylar C Cook
  • Mark Pogson
  • Sai T Reddy

Abstract

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

Suggested Citation

  • Ulrike Menzel & Victor Greiff & Tarik A Khan & Ulrike Haessler & Ina Hellmann & Simon Friedensohn & Skylar C Cook & Mark Pogson & Sai T Reddy, 2014. "Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing," PLOS ONE, Public Library of Science, vol. 9(5), pages 1-12, May.
    • Handle: RePEc:plo:pone00:0096727
    DOI: 10.1371/journal.pone.0096727
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    1. Shuo Li & Marie-Paule Lefranc & John J. Miles & Eltaf Alamyar & Véronique Giudicelli & Patrice Duroux & J. Douglas Freeman & Vincent D. A. Corbin & Jean-Pierre Scheerlinck & Michael A. Frohman & Paul , 2013. "IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling," Nature Communications, Nature, vol. 4(1), pages 1-13, December.
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