title: Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.
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.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Menzel
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Victor
authors.initials: V
authors.lastname: Greiff
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Tarik A
authors.initials: TA
authors.lastname: Khan
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Haessler
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Ina
authors.initials: I
authors.lastname: Hellmann
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Friedensohn
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Skylar C
authors.initials: SC
authors.lastname: Cook
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Mark
authors.initials: M
authors.lastname: Pogson
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Sai T
authors.initials: ST
authors.lastname: Reddy
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D019295:Computational Biology; D005260:Female; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D051379:Mice; D016133:Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D000906:Antibodies