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A Thesis: Entitled Improving a Method for High A Thesis: entitled Improving a Method for High-Resolution HLA-Typing In Transplantation by Afnan Sami H Malebari Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Sciences: Medical Microbiology and Immunology _________________________________________ Dr. Stanislaw Stepkowski, Committee Chair _________________________________________ Dr. Kevin Pan, Committee Member _________________________________________ Dr. Sadik A. Khuder, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2017 Copyright 2017, Afnan Sami H Malebari This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Improving a Method for High-Resolution HLA-Typing In Transplantation by Afnan Sami H Malebari Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Sciences: Medical Microbiology and Immunology The University of Toledo May 2017 For a successful unrelated organ transplantation with low risk of graft-versus-host disease and mortality, it is important to have a close human leukocyte antigen (HLA) between donor and recipient. In other words, in diagnostic laboratories, HLA typing and antibody profile are the key rules to test donor and recipients compatibility. However, due to that HLA genes are the most polymorphic genes in the human genome, high- resolution HLA typing is challenging by using well-tried methods. Sanger sequencing- based typing (SBT) is the gold standard method in sequencing and continuously identify new HLA alleles. Even though, SBT has disadvantageous as a result of its confined sensitivity and its failure to perform parallel sequencing of multiple targets. In the other hand, next generation sequencing (NGS) technologies may improve the sequencing process for HLA typing because it allows massively parallel sequencing, rapid, and cost- effective than SBT. So, NGS is a promising tool to overcome intrinsic HLA typing problems. iii Therefore, in this study, I am improving a preliminary assay by NGS for high- resolution HLA typing in transplantation. The preliminary assay was designed for high- resolution HLA typing for class-I and class-II HLA has some obstacles in identifying four-digit HLA alleles, but I will focus on improving class-I HLA typing and work on model solutions for HLA class-I (HLA-A, B, and C) then apply it on class-II. The preliminary assay library preparation has two different types of primers; broad primer pairs to increase the amplification ratio of the target genes than its pseudogenes and target specific primer pairs to amplify specific regions of the target genes. However, the redesigned broad primers for class-I HLA genes were able to amplify more specifically the target genes and reduced the amplification of pseudogenes than the previously designed broad primers. Additionally, a preliminary computer program plays a vital role in analyzing the sequence and identifying the correct high-resolution HLA alleles. The ability of the right allele calls increased by excluding any sequence read length less than 90% of the expected sequence read length of each amplicon, and minimizing interference sequence reads by redesigning broad primers. iv To my husband, Shadi Melebari, your presence in my life, your love, your standing beside me encouraged me to overcome difficulties of this journey. I am forever thankful of the enormous sacrifices you have made along the way. To my Mom and Dad, Thuraya Melebari and Sami Melebari, I could never have done this without your, encouragement to be the best in my studies and confidence and you always taught me the love of knowledge, the determination and patience. Thank you for teaching me to believe in myself, in Allah, and in my dreams. Acknowledgements I would like to express my appreciation and gratitude to my advisor, Dr. Stepkowski for his support, expert guidance, and advice through my Master studies. I would also like to thank the faculty and staff in the department of Medical microbiology and immunology at the University of Toledo. To those who help me in my research and light the darkness that sometimes stand in my way. To those who provided me with assistance, ideas and information, possibly without feeling in turn. So they have the thanks of me all, and particularly of them; Caitlin Baum, Dr.Beata Mierzejewska, Dr.Willey James, Dulat Bekbolsynov and Erin Crawford. A special gratitude I give to Dr. Thomas Blomquist, whose contribution in stimulating suggestions and modifying the computer program, helped me to complete my project. vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ....................................................................................................... xiii List of Symbols ................................................................................................................ xiv 1 Chapter 1 ..................................................................................................................1 1.1 Introduction .......................................................................................................... 1 1.2 Objectives ........................................................................................................... 13 2 Chapter 2 ................................................................................................................... 16 2.1 Literature Survey ................................................................................................ 16 3 Chapter 3 ................................................................................................................... 21 3.1 Materials and Methods ....................................................................................... 21 3.1.1 Samples ....................................................................................................... 21 vii 3.1.2 DNA extractions ......................................................................................... 21 3.1.3 DNA concentration and purity determination ............................................. 22 3.1.4 Reagent Design ........................................................................................... 23 3.1.5 Library construction .................................................................................... 24 3.1.6 Next Generation sequencing ....................................................................... 27 3.1.7 Evaluating primers and PCR reaction products .......................................... 28 3.1.8 Data processing ........................................................................................... 28 3.1.9 Analyzing the raw sequence data without the software. ............................. 29 3.1.10 Statistical Analysis. ..................................................................................... 30 4 Chapter 4 ................................................................................................................... 33 4.1 Results ................................................................................................................ 33 4.1.1 Obstacles in the preliminary assay. ............................................................. 33 4.1.2 Redesigning broad primers. ........................................................................ 39 4.1.3 Performance testing of competitive amplicon library preparation. ............ 56 4.2 Discussion .......................................................................................................... 72 5 Chapter 5 ................................................................................................................... 81 5.1 Conclusion .......................................................................................................... 81 Reference .......................................................................................................................... 83 A Preliminary assay primers sequences ............................................................... 91 viii List of Tables Table 3.1. HLA Class-I-broad primer sets. ....................................................................... 31 Table 3.2. HLA Class-I target specific primer sets. .......................................................... 32 Table 4.1. Average percentage of preliminary NGS assay of sequence reads agreement with Sanger sequence reads per amplicon among 6 patients .................... 38 Table 4.2. Redesigned broad primers for HLA-A exon 4................................................. 40 Table 4.3. Redesigned broad primers for HLA-B exon 2, 3, and 4. ................................. 46 Table 4.4. Redesigned broad primers for HLA-C exon
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