The Korean Journal of Blood Transfusion (대한수혈학회지)

The Korean Society of Blood Transfusion (대한수혈학회)
권호 표지
DOI QR코드

DOI QR Code

Development of HLA-A, -B and -DR Typing Method Using Next-Generation Sequencing

차세대염기서열분석법을 이용한 HLA-A, -B 그리고 -DR 형별 분석법 개발

  • Received : 2018.04.23
  • Accepted : 2018.11.07
  • Published : 2018.12.31
⟨ Previous Next ⟩

Abstract

Background: Research on next-generation sequencing (NGS)-based HLA typing is active. To resolve the phase ambiguity and long turn-around-time of conventional high resolution HLA typing, this study developed a NGS-based high resolution HLA typing method that can handle large-scale samples within an efficient testing time. Methods: For HLA NGS, the condition of nucleic acid extraction, library construction, PCR mechanism, and HLA typing with bioinformatics were developed. To confirm the accuracy of the NGS-based HLA typing method, the results of 192 samples HLA typed by SSOP and 28 samples typed by SBT compared to NGS-based HLA-A, -B and -DR typing. Results: DNA library construction through two-step PCR, NGS sequencing with MiSeq (Illumina Inc., San Diego, USA), and the data analysis platform were established. NGS-based HLA typing results were compatible with known HLA types from 220 blood samples. Conclusion: The NSG-based HLA typing method could handle large volume samples with high-throughput. Therefore, it would be useful for HLA typing of bone marrow donation volunteers.

배경: 최근 차세대염기서열분석법(Next Generation Sequencing: NGS)을 이용한 HLA 형별 분석에 대한 연구가 활발히 진행되고 있다. 이에 HLA 고해상도 분석법의 내재적 한계인 위상 모호성의 문제를 해결하고, 대량 검체 처리가 가능한 NGS 기반 고해상도 HLA 형별 검사법을, 자체 기술로 개발하고자 본 연구를 실시하였다. 방법: HLA NGS를 위한 핵산 추출 조건, 라이브러리 제작 및 PCR 체계 확립, 그리고 생물정보학을 이용한 HLA 형별 분석법을 개발하였다. 본 기관에서 개발한 NGS 기반 HLA 형별 검사의 정확성을 알아보기 위해 SSOP법으로 HLA 형별을 알고 있는 192개 검체와 SBT법으로 HLA 형별을 알고 있는 28개 검체에 대해 NGS 기반으로 검사한 HLA-A, -B 그리고 -DR 형별 결과를 비교해 보았다. 결과: 두 단계의 PCR을 통한 DNA 라이브러리 제작과 MiSeq (Illumina Inc., San Diego, USA) 기기를 이용한 NGS 시퀸싱 그리고 데이터 분석 시스템을 구축하였다. 기존에 HLA 형별을 알고 있는 220개 혈액 검체에 대해 NGS 기반 HLA 형별검사 결과가 모두 일치함을 확인하였다. 결론: NSG 기반 HLA 형별 검사법은 많은 검체를 효율적인 시간 내에 처리가 가능하여 조혈모세포기증 희망자 HLA 검사 등에 유용할 것으로 기대된다.

Keywords

Acknowledgement

Supported by : 중소기업청

References

  1. Guy K, Krajewski AS, Dewar AE. Expression of MHC class II antigens in human B-cell leukemia and non-Hodgkin's lymphoma. Br J Cancer 1986;53:161-73 https://doi.org/10.1038/bjc.1986.31
  2. Kim HJ. HLA mismatch allogeneic blood and marrow transplantation. Korean J Med 2014;86:1-13 https://doi.org/10.3904/kjm.2014.86.1.1
  3. Kim MH, Choi SE, Oh HB. Human leukocyte antigen typing proficiency surveys in Korea, 2005-2006. Korean J Lab Med 2007;27:442-50 https://doi.org/10.3343/kjlm.2007.27.6.442
  4. Lee KW, Park MH. A comparative study of HLA-DR6 by serology and PCR-SSOP typing. Korean J Clin Pathol 1994;14:109-22
  5. Cao K, Chopek M, Fernandez-Vina MA. High and intermediate resolution DNA typing system for class I HLA-A, B, C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Rev Immunogenet 1999;1:177-208
  6. Cha CH, Kim MH, Chung HJ, Choi SE, Oh HB. Evaluation of BioSewoomTM HLA-A,-B,-C PCR/SSP kit. Korean J Lab Med 2007;27:360-8 https://doi.org/10.3343/kjlm.2007.27.5.360
  7. In JW, Roh EY, Oh SH, Shin S, Park KU, Song EY. Allele and haplotype frequencies of Human leukocyte antigen-A, -B,-C,-DRB1, and -DQB1 from sequence-based DNA typing data in Koreans. Ann Lab Med 2015;35:429-35 https://doi.org/10.3343/alm.2015.35.4.429
  8. Park YJ, Kim HS, Yoon CE, Kwon OJ, Kim YS. Resolution of ambiguous HLA genotyping in Korean by multi-group-specific sequence-based typing. Yonsei Med J 2014;55:1005-13 https://doi.org/10.3349/ymj.2014.55.4.1005
  9. Petersdorf EW, Gooley TA, Anasetti C, Martin PJ, Smith AJ, Mickelson EM, et al. Optimizing outcome after unrelated marrow transplantation by comprehensive matching of HLA class I and II alleles in the donor and recipient. Blood 1998;92:3515-20 https://doi.org/10.1182/blood.V92.10.3515
  10. Wang C, Krishnakumar S, Wilhelmy J, Babrzadeh F, Stepanyan L, Su LF, et al. Highthroughput, high-fidelity HLA genotyping with deep sequencing. Proc Natl Acad Sci U S A 2012;109:8676-81 https://doi.org/10.1073/pnas.1206614109
  11. Lange V, Bohme I, Hofmann J, Lang K, Sauter J, Shone B, et al. Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing. BMC Genomics 2014;15:63 https://doi.org/10.1186/1471-2164-15-63
  12. Gabriel C, Furst D, Fae I, Wenda S, Zollikofer C, Mytilineos J, et al. HLA typing by nextgeneration sequencing-getting closer to reality. Tissue Antigens 2014;83:65-75 https://doi.org/10.1111/tan.12298
  13. Gandhi MJ, Ferriola D, Huang Y, Duke JL, Monos D. Targeted next-generation sequencing for human leukocyte antigen typing in a clinical laboratory: metrics of relevance and considerations for its successful implementation. Arch Pathol Lab Med 2017;141:806-12 https://doi.org/10.5858/arpa.2016-0537-RA
  14. Profaizer T, Coonrod EM, Delgado JC, Kumanovics A. Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq. Hum Immnol 2015;76:897-902 https://doi.org/10.1016/j.humimm.2015.08.002
  15. Pirooznia M, Kramer M, Parla J, Goes FS, Potash JB, McCombie WR, et al. Validation and assessment of variant calling pipelines for next-generation sequencing. Hum Genomics 2014;8:14 https://doi.org/10.1186/1479-7364-8-14
  16. Kim JH, Park WY, Kim NKD, Jang SJ, Chun SM, Sung CO, et al. Good laboratory standards for clinical next-generation sequencing cancer panel tests. J Pathol Transl Med 2017;51:191-204 https://doi.org/10.4132/jptm.2017.03.14
  17. Seo DH, Cho DY, Kim JH, Kim SY, Cho SE, Oh M. Clinical study of non-invasive prenatal testing using next-generation sequencing. J Lab Med Qual Assur 2015;37:214-18 https://doi.org/10.15263/jlmqa.2015.37.4.214
  18. Gargis AS, Kalman L, Lubin IM. Assuring the quality of next-generation sequencing in clinical microbiology and public health laboratories. J Clin Mirobiol 2016;54:2857-65 https://doi.org/10.1128/JCM.00949-16
  19. Na HS. Performance comparison of benchtop next-generation sequencing systems. J Bacteriol Virol 2014;44:208-13 https://doi.org/10.4167/jbv.2014.44.2.208
  20. Yohe S, Thyagarajan B. Review of clinical next-generation sequencing. Arch Pathol Lab Med 2017;141:1544-57 https://doi.org/10.5858/arpa.2016-0501-RA
  21. Yin Y, Lan JH, Nguyen D, Valenzuela N, Takemura P, Bolon YT, et al. Application of high-throughput next-generation sequencing for HLA typing on buccal extracted DNA: results from over 10,000 donor recruitment samples. PLoS One 2016;11:e0165810 https://doi.org/10.1371/journal.pone.0165810
  22. Kunkel M, Duke J, Ferriola D, Lind C, Monos A. Molecular methods for human leukocyte antigen typing: current practices and future directions. In: Detrick B, Hamilton RG, Schmitz JL. Manual of molecular and clinical laboratory immunology. 8th ed. Washington, DC: ASM, 2016