naa a22 4500 528784455 20180924065517.0 cr unu---uuuuu 180924e20140508xx s 000 0 eng 10.3929/ethz-b-000086651 doi 10.1371/journal.pone.0096727 doi (ETHRESEARCH)oai:www.research-collection.ethz.ch:20.500.11850/86651 Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing [Elektronische Daten] [Ulrike Menzel, Victor Greiff, Tarik A. Khan, Ulrike Haessler, Ina Hellmann, Simon Friedensohn, Skylar C. Cook, Mark Pogson, Sai T. Reddy] PLoS ONE Open access ethresearch 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. Creative Commons Attribution 4.0 International http://creativecommons.org/licenses/by/4.0 ethresearch Menzel Ulrike joint author Greiff Victor joint author Khan Tarik A. joint author Haessler Ulrike joint author Hellmann Ina joint author Friedensohn Simon joint author Cook Skylar C. joint author Pogson Mark joint author Reddy Sai T. joint author PLoS ONE S.l. : Public Library of Science 9 (5), p. e96727 1932-6203 http://hdl.handle.net/20.500.11850/86651 text/html WWW-Backlink auf das Repository (Open access) 1 Journal Article ethresearch ETHRESEARCH 856 40 http://hdl.handle.net/20.500.11850/86651 text/html WWW-Backlink auf das Repository (Open access) ETHRESEARCH 700 1- Menzel Ulrike joint author ETHRESEARCH 700 1- Greiff Victor joint author ETHRESEARCH 700 1- Khan Tarik A. joint author ETHRESEARCH 700 1- Haessler Ulrike joint author ETHRESEARCH 700 1- Hellmann Ina joint author ETHRESEARCH 700 1- Friedensohn Simon joint author ETHRESEARCH 700 1- Cook Skylar C. joint author ETHRESEARCH 700 1- Pogson Mark joint author ETHRESEARCH 700 1- Reddy Sai T. joint author ETHRESEARCH 773 0- PLoS ONE S.l. : Public Library of Science 9 (5), p. e96727 1932-6203 BK010053 XK010053 XK010000 ETHRESEARCH ETHRESEARCH ETHRESEARCH Journal Article Open access