A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping.

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.

[1]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[2]  A. Hughes,et al.  Natural selection at major histocompatibility complex loci of vertebrates. , 1998, Annual review of genetics.

[3]  R Higuchi,et al.  High-resolution, high-throughput HLA genotyping by next-generation sequencing. , 2009, Tissue antigens.

[4]  J J Goedert,et al.  Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS. , 2001, The New England journal of medicine.

[5]  S. Adams,et al.  Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification , 2004, Journal of Translational Medicine.

[6]  Matthew S. Cook,et al.  Immunodominant Tuberculosis CD8 Antigens Preferentially Restricted by HLA-B , 2007, PLoS pathogens.

[7]  Yao Ju,et al.  HLA-B~*5701 screening for hypersensitivity to abacavir , 2011 .

[8]  H. Inoko,et al.  The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. , 2002, Blood.

[9]  D. Cook,et al.  Resolution of cis-trans ambiguities between HLA-DRB1 alleles using single-strand conformation polymorphisms and sequencing. , 2001, Tissue antigens.

[10]  P. Hedrick.  PATHOGEN RESISTANCE AND GENETIC VARIATION AT MHC LOCI , 2002, Evolution; international journal of organic evolution.

[11]  S. Ringquist,et al.  Multiplex HLA-typing by pyrosequencing. , 2009, Methods in molecular biology.

[12]  F. Marincola,et al.  High Throughput Hla Sequence-Based Typing (Sbt) Utilizing the Abi Prism® 3700 Dna Analyzer , 2001, Tumori.

[13]  M. Horowitz,et al.  HLA-A disparities illustrate challenges for ranking the impact of HLA mismatches on bone marrow transplant outcomes in the United States. , 2009, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[14]  Andrew J. McMichael,et al.  Common West African HLA antigens are associated with protection from severe malaria , 1991, Nature.

[15]  A. Sanchez,et al.  Sequence-Based Human Leukocyte Antigen—B Typing of Patients Infected with Ebola Virus in Uganda in 2000: Identification of Alleles Associated with Fatal and Nonfatal Disease Outcomes , 2007, The Journal of infectious diseases.

[16]  S. E. Marsh.  Nomenclature for factors of the HLA system, update February 2010. , 2010, Tissue antigens.

[17]  T. Harkins,et al.  Major histocompatibility complex genotyping with massively parallel pyrosequencing , 2009, Nature Medicine.

[18]  M. Hentze,et al.  A Perfect Message RNA Surveillance and Nonsense-Mediated Decay , 1999, Cell.

[19]  M. Bunce,et al.  Molecular HLA typing--the brave new world. , 1997, Transplantation.

[20]  James Robinson,et al.  The IMGT/HLA database , 2008, Nucleic Acids Res..

[21]  F. Balloux,et al.  Pathogen-Driven Selection and Worldwide HLA Class I Diversity , 2005, Current Biology.

[22]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[23]  R. Knight,et al.  Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex , 2008, Nature Methods.

[24]  David Heckerman,et al.  Statistical Resolution of Ambiguous HLA Typing Data , 2008, PLoS Comput. Biol..

[25]  W. Andiman,et al.  Virologic and Host Characteristics of Human Immunodeficiency Virus Type 1-Infected Pediatric Long Term Survivors , 2006, The Pediatric infectious disease journal.

[26]  C. Chu,et al.  Discovery of HLA‐DRB1*0331 in a Taiwanese marrow donor and the importance of sequence‐based typing in a rare or previously unrecognized allele , 2007, International journal of immunogenetics.

[27]  J. Rothberg,et al.  The development and impact of 454 sequencing , 2008, Nature Biotechnology.

[28]  C. Chu,et al.  Detection of a rare Caucasoid HLA‐DRB1*0337 in a Taiwanese bone marrow donor using sequence‐based typing method , 2010, International journal of immunogenetics.

[29]  E. Thorsby,et al.  HLA associated genetic predisposition to autoimmune diseases: Genes involved and possible mechanisms. , 2005, Transplant immunology.

[30]  Helene Polin,et al.  Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification. , 2009, Human immunology.

[31]  C. Tukuitonga,et al.  Low-cost, simultaneous, single-sequence genotyping of the HLA-A, HLA-B and HLA-C loci. , 2006, Tissue Antigens.

[32]  K. Ghosh,et al.  Implications of HLA sequence-based typing in transplantation. , 2008, Journal of postgraduate medicine.

[33]  U. Stenzel,et al.  Parallel tagged sequencing on the 454 platform , 2008, Nature Protocols.

[34]  M. Franceschi,et al.  HLA-A*01 is Associated with Late Onset of Alzheimer's Disease in Italian Patients , 2009, International journal of immunopathology and pharmacology.

[35]  James Robinson,et al.  IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex , 2003, Nucleic Acids Res..

[36]  A. Zachary,et al.  Immunogenetics and immunology in transplantation , 2010, Immunologic research.

[37]  T. Zhao,et al.  Identification of a new HLA allele, A*1114, in a Chinese family. , 2003, Tissue antigens.