University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2017 Next Generation Sequencing-Based Genotyping of Human Blood Groups: FY, JK and ABO Genes Altayar, Malik Abdullah http://hdl.handle.net/10026.1/9325 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. Next Generation Sequencing-Based Genotyping of Human Blood Groups: FY, JK and ABO Genes by Malik Abdullah Altayar A thesis submitted to Plymouth University in partial fulfilment for the degree of DOCTOR OF PHILOSOPHY School of Biomedical and Healthcare Sciences September 2016 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author's prior consent. I Acknowledgements First of all, I thank God for all his blessings that enable me to complete this PhD project. My sincere gratitude is to my parents and all my family for their great support. I am sincerely grateful for the support of my wife and daughters. I would like to express my deepest gratitude to Tabuk University for funding my studies and providing great support in my scholarship in the United Kingdom. I would like to express my special appreciation to my supervisors, Professor Neil Avent and Dr. Tracey Madgett for their endless support and guidance. It has been a great pleasure and honour to be part of their research team. I would like to thank Dr. Michele Kiernan, Dr. Kris Jeremy and Dr. Paul Waines for their valuable help. Finally, I would like to thank all my friends and anyone who have been supporting me throughout my scholarship. II AUTHOR'S DECLARATION At no time during the registration for the degree of Doctor of Philosophy has the author been registered for any other University award without prior agreement of the Graduate Sub-Committee. Name: Malik Abdullah Altayar Signature: Date: Word count of the thesis’s main body: 64,612 words III Award Margaret Kenwright Young Scientist Award 2016 from the British Blood Transfusion Society (BBTS) for the following work: Altayar, M.A., Madgett, T.E., Kiernan, M., Halawani, A.J., Avent, N.D. (2016). Next generation sequencing of JK (SLC14A1) gene reveals higher frequency of variant alleles, novel allele-defining SNPs (allele reference fingerprints) and reassignment of a purported JKnull allele. Transfusion Medicine, 26, suppl. 2, 3-24. Publications N. D. Avent, T. E. Madgett, A. J. Halawani, M. A. Altayar, M. Kiernan, A. J. Reynolds, & X. Li. (2015). Next-generation sequencing: academic overkill or high-resolution routine blood group genotyping? ISBT Science Series, 10, suppl. 1, 250-256. IV Conference participation M. Altayar, A. Halawani, M. Kiernan, A. Reynolds, N. Kaushik, T. Madgett & N. Avent (2013) Next Generation Sequencing of ABO, Duffy and Kidd Blood Group Genotyping. Transfusion Medicine, 23, suppl. 2; 30-71. A. J. Halawani, Altayar, M. A., M. Kiernan, N. Kaushik, A. Reynolds, T. Madgett & N. Avent (2013) Comprehensive Genotyping for Kell and Rh Blood Group Systems by Next-generation DNA Sequencing. Transfusion Medicine 23, suppl. 2; 30-71. Altayar, M. A., Halawani, A. J., Kiernan, M., Reynolds, A. J., Kaushik, N., Madgett, T. E. & Avent, N. D. (2014). Extensive Genotyping of Blood Groups Duffy, Kidd and ABO by Next-generation Sequencing. Vox Sang, 107, suppl. 1, 57-248. Halawani, A. J., Altayar, M. A., Kiernan, M., Reynolds, A. J., Kaushik, N., Madgett, T. E. & Avent, N. D. (2014). Can Next-generation DNA Sequencing Solve the RH Complexity for Genotyping? Vox Sang, 107, suppl. 1, 57-248. Altayar, M. A., Halawani, A. J., Kiernan, M., Madgett, T. E. & Avent, N. D. (2015). Complete Gene Sequencing of ABO Blood Group by Next-generation Sequencing. Vox Sang, 109, suppl. 1, 1-379. Halawani, A. J., Altayar, M. A., Kiernan, M., Li, X., Madgett, T. E. & Avent, N. D. (2015). High Resolution Genotyping of the Rh Blood Group System by Next-generation Sequencing Vox Sang, 109, suppl. 1, 1-379. Abstracts are shown in Appendix A V Abstract Next Generation Sequencing-Based Genotyping of Human Blood Groups: FY, JK and ABO Genes. Malik Abdullah Altayar Serological discrepancies in matching blood group antigens between donors and patients for blood transfusion may lead to alloimmunisation, especially in multiply transfused patients. Blood group genotyping (BGG) has contributed in reducing this issue. ABO, Fy and Jk antigens are among those to be causative for alloimmunisation through transfusion or pregnancy. The number of alleles of these clinically significant blood groups is ever increasing. Currently, all commercially available high-throughput BGG platforms are only based on pre-defined polymorphisms. Consequently, novel or rare alleles that might have clinical significance are not identified. Next generation sequencing (NGS) circumvents this issue by providing high-throughput comprehensive genotyping of blood group genes in discovery mode to find all existing and novel mutations. Accordingly, a large number of individuals can be genotyped in a single run. Here, we describe an NGS-based method coupled with long-range polymerase chain reaction (LR-PCR) for high-throughput, rapid and extensive genotyping of FY, JK and ABO blood group genes. The Ion Torrent Personal Genome Machine (PGMTM) was used for sequencing the entire FY, JK and ABO blood group genes including flanking regions. Accordingly, high resolution genotyping was obtained. 53 genomic DNA samples were sequenced and genotyped for FY, 67 for JK and 47 for ABO. Sequencing data were aligned to the gene reference sequence derived from the human genome (hg19) to analyse variants. Analysis was accomplished by software packages, such as Ion Torrent SuiteTM plugins. Sanger sequencing of cDNA and cDNA clones was used to confirm findings in the JK gene. The sequencing data had a coverage depth of more than 5000x for FY, 700x for JK and 600x for ABO. NGS data matched with the serological phenotypes of FY alleles FY*A, FY*B and FY*02 Null main polymorphisms, such as FY*A/FY*B (125G>A) in exon 2 and (-67 T>C) in the promotor region. JK variant analysis revealed that the JK*01W.01 allele (130G>A) is common (10/67 samples) with normal antigenicity. The previously described silencing polymorphism (810G>A), leading to a purported JK*B null allele, restores a splice site and does not correlate with loss of Jkb antigenicity (10/67 samples). JK intron analysis revealed several new JK alleles described in this thesis. All 7 exons, introns and the flanking regions of the ABO gene were covered by only four amplicons. Several rare O alleles were found, such as O73 and O75, while one suggested novel O allele was characterised by a missense SNP 482G>A (Arg161His) in exon 7. The ABO reference sequence from hg19 appeared to resemble (O01 and O02) alleles. The intronic SNPs might be used to distinguish between alleles more accurately as a correlation of the intronic SNPs with the alleles was noted for the homozygous O alleles. It is predicted that NGS-based genotyping will replace not only microarray-based genotyping but also serology in the blood group typing of individuals, with great advancements in technology and molecular knowledge being expected in the near future. VI Table of Contents Chapter 1: Introduction 1 1.1 Introduction to blood groups 1 1.2 Blood group terminology 2 1.3 The molecular basis of blood group polymorphisms 6 1.4 The immunogenicity of blood groups 10 1.4.1 Haemolytic transfusion reaction (HTR) 10 1.4.2 Haemolytic disease of fetus and newborn (HDFN) 11 1.5 ABO blood group system 12 1.5.1 Discovery 13 1.5.2 Biosynthesis of ABO blood group antigens 15 1.5.3 Inheritance and molecular genetics 18 1.5.4 ABO polymorphisms (ABO alleles) 21 1.5.5 ABO antibodies (clinical significance) 24 1.6 Duffy blood group system (FY) 25 1.6.1 Duffy glycoprotein and FY gene. 25 1.6.2 Molecular basis of FY blood group system 27 1.6.3 FY antibodies (clinical significance) 31 1.7 Kidd blood group system (JK) 31 1.7.1 JK glycoprotein and the JK gene 32 1.7.2 Molecular basis of JK blood group system 34 1.7.3 JK antibodies (clinical significance) 35 1.8 Blood group genotyping 36 1.8.1 Serology and genotyping 36 1.8.2 Applications of BGG 38 1.8.3 High throughput 39 1.8.4 Genotyping technology and methodology 40 1.8.5 Next-generation sequencing 45 1.8.6 Ion Torrent Personal Genome MachineTM (Ion PGMTM) 49 1.8.7 NGS and BGG 54 1.9 Thesis aims 56 CHAPTER 2: Materials and Methods 58 2.1 Genomic DNA (gDNA) and RNA from whole blood samples 58 2.2 Sequencing of blood group genes by next-generation sequencing 58 2.2.1 gDNA extraction and purification 61 2.2.2 RNA extraction and purification 62 2.2.2.1 RNA quantification 64 2.2.3 DNA quantitation 65 2.2.4 Amplicon library preparation for next-generation sequencing 66 2.2.4.1 Primers 66 2.2.4.2 LR-PCR amplicon amplification 70 2.2.4.3 Agarose gel electrophoresis 72 2.2.4.4 Purification 74 VII 2.2.4.5 Sample aliquots 77 2.2.4.6 Amplicon library fragmentation (purified fragmented library) 78 2.2.4.7 The Agilent® 2100 Bioanalyzer 80 2.2.4.8 Optimisations of the fragmentation reaction time 83 2.2.4.9 Ligation of barcoded adapters (purified-ligated library) 87 2.2.4.10 Size selection of the library 90 2.2.4.11.