HLA Typing pp 135-153 | Cite as

High-Resolution Full-Length HLA Typing Method Using Third Generation (Pac-Bio SMRT) Sequencing Technology

  • Sheetal Ambardar
  • Malali GowdaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1802)


The human HLA genes are among the most polymorphic genes in the human genome. Therefore, it is very difficult to find two unrelated individuals with identical HLA molecules. As a result, HLA Class I and Class II genes are routinely sequenced or serotyped for organ transplantation, autoimmune disease-association studies, drug hypersensitivity research, and other applications. However, these methods were able to give two or four digit data, which was not sufficient enough to understand the completeness of haplotypes of HLA genes. To overcome these limitations, we here described end-to-end workflow for sequencing of HLA class I and class II genes using third generation sequencing, SMRT technology. This method produces fully-phased, unambiguous, allele-level information on the PacBio System.


Single molecule Real time Sequencing HLA typing PacBio High resolution Full length 



We are thankful to Dr. Sudhir Krishna, NCBS for his scientific guidance and funding this research. We thank Dr. Latha Jagannathan and Dr. Nutan Dighe, Bangalore Medical Services Trust, Bangalore for their constant support and providing the samples for research work. We thank Mohammad Zahid from Shiva Scientific/GenDx for providing PacBio HLA primers. Thanks to Dr. Anil Singh and Mr. Mohit from Institute of Himalayan Bioresource Technology, Palampur and also thanks to Paras Yadav, Imperial Life Science (P) LTD., Delhi for their help to access PacBio sequencer. We appreciate Centre for Cellular and Molecular Platforms and Department of Biotechnology, Government of India for their support for this project.


  1. 1.
    Carrington M, O’Brien SJ (2003) The influence of HLA genotype on AIDS. Annu Rev Med 54:535–551CrossRefPubMedGoogle Scholar
  2. 2.
    Shina T, Hosomichi K, Inoko H, Kulski JK (2009) The HLA genomic loci map: expression, interaction, diversity and disease. J Hum Genet 5:15–39CrossRefGoogle Scholar
  3. 3.
    De Santis D, Dinauer D, Duke J, Erlich HA, Holcomb CL et al (2013) 16th IHIW: review of HLA typing by NGS. Int J Immunogenet 40:72–76CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Morishima Y, Sasazuki T, Inoko H, Juji T, Akaza T, Yamamoto K et al (2002) Matched unrelated donors. Blood 99:4200–4206CrossRefPubMedGoogle Scholar
  5. 5.
    Horton R, Gibson R, Coggill P, Miretti M, Allcock RJ, Almeida J et al (2008) Variation analysis and gene annotation of eight MHC haplotypes: the MHC Haplotype Project. Immunogenetics 60:1–18CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lind C, Ferriola D, Mackiewicz K, Heron S, Rogers M, Slavich L et al (2010) Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing. Hum Immunol 71:1033–1042CrossRefPubMedGoogle Scholar
  7. 7.
    Bentley G, Higuchi R, Hoglund B, Goodridge D, Sayer D, Trachtenberg EA, Erlich HA (2009) High-resolution, high-throughput HLA genotyping by next generation sequencing. Tissue Antigens 74:393–403CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Enrich RL, Jia X, Anderson S, Banks E, Gao X et al (2011) Next generation sequencing for HLA typing of class I loci. BMC Genomics 12:42CrossRefGoogle Scholar
  9. 9.
    Moonsamy PV, Williams T, Bonella P, Holcomb CL, Höglund BN et al (2013) High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ system for simplified amplicon library preparation. Tissue Antigens 81:141–114CrossRefPubMedGoogle Scholar
  10. 10.
    Gowda M, Ambardar S, Dighe N, Manjunath A, Shankaralingu C, Hallappa P, Harting J, Ranade S, Jagannathan L, Krishna S (2016) Comparative analyses of low, medium and high-resolution HLA typing technologies. J Clin Cell Immunol 7(2).
  11. 11.
    Nelson WC, Pyo CW, Vogan D, Wang R, Pyon YS et al (2015) An integrated genotyping approach for HLA and other complex genetic systems. Hum Immunol 76:928–938CrossRefPubMedGoogle Scholar
  12. 12.
    Mayor NP, Robinson J, McWhinnie AJM, Ranade S, Eng K et al (2015) HLA typing for the next generation. PLoS One 10:e0127153CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    QIAGEN (2016) QIAamp® DNA mini and blood mini handbook (cat. no. 51304). Accessed 10 Dec 2017
  14. 14.
    GenDX (2017) NGSgo-AmpX instruction for use (cat. no. 2341102 & 2341502). Accessed 10 Dec 2017
  15. 15.
    Pacific Bioscience (2016) HLA getting started guide (cat. no. 100-259-100). Accessed 10 Dec 2017Google Scholar
  16. 16.
    Pacific Bioscience (2015) Template preparation and sequencing guide (cat. no. 100-259-100). Accessed 10 Dec 2017Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre for Functional Genomics and BioinformaticsInstitute of TransDisciplinary Health Sciences and TechnologyBangaloreIndia

Personalised recommendations