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Characterization of Immunoglobulin Repertoires after Vaccination in OmniRatTM and Monoclonal CD5+ B Cell Expansion in A20BKOsCYLDBOE Mice using an Ion Torrent PGM High-Throughput Sequencing Platform

  • With the advent of high­throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. The dissection of IG repertoires promises to transform our understanding of the adaptive immune system dynamics. Advances in sequencing technology now also allow the use of the Ion Torrent Personal Genome Machine (PGM) to cover the full length of IG mRNA transcripts. The applications of this bench­top scale HTS platform range from identification of new therapeutic antibodies to the deconvolution of malignant B cell tumors. In the context of this thesis, the usability of the PGM is assessed to investigate the IG heavy chain (IGH) repertoires of animal models. First, an innovate bioinformatics approach is presented to identify antigendriven IGH sequences from bulk sequenced bone marrow samples of transgenic humanized rats, expressing a human IG repertoire (OmniRatTM). We show, that these rats mount a convergent IGH CDR3 response towards measles virus hemagglutinin protein and tetanus toxoid, with high similarity to human counterparts. In the future, databases could contain all IGH CDR3 sequences with known specificity to mine IG repertoire datasets for past antigen exposures, ultimately reconstructing the immunological history of an individual. Second, a unique molecular identifier (UID) based HTS approach and network property analysis is used to characterize the CLL­like CD5+ B cell expansion of A20BKO mice overexpressing a natural short splice variant of the CYLD gene (A20BKOsCYLDBOE). We could determine, that in these mice, overexpression of sCYLD leads to unmutated subvariant of CLL (U­CLL). Furthermore, we found that this short splice variant is also seen in human CLL patients highlighting it as important target for future investigations. Third, the UID based HTS approach is improved by adapting it to the PGM sequencing technology and applying a custom­made data processing pipeline including the ImMunoGeneTics (IMGT) database error detection. Like this, we were able to obtain correct IGH sequences with over 99.5% confidence and correct CDR3 sequences with over 99.9% confidence. Taken together, the results, protocols and sample processing strategies described in this thesis will improve the usability of animal models and the Ion Torrent PGM HTS platform in the field if IG repertoire research.

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Author:Jean-Philippe Bürckert
Referee:Claude P. Muller, Jobst Meyer
Advisor:Claude P. Muller
Document Type:Doctoral Thesis
Date of completion:2019/02/18
Publishing institution:Universität Trier
Granting institution:Universität Trier, Fachbereich 1
Date of final exam:2018/12/18
Release Date:2019/03/21
GND Keyword:Immunoglobulin; Immunsystem; Messenger-RNS; Repertoire; Sequenzanalyse; Tiermodell
Number of pages:134
Institutes:Fachbereich 1
Dewey Decimal Classification:0 Informatik, Informationswissenschaft, allgemeine Werke / 00 Informatik, Wissen, Systeme / 004 Datenverarbeitung; Informatik
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCC BY-SA: Creative-Commons-Lizenz 4.0 International

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