Evaluation of a novel ultra-high-resolution assay and analytic software for next generation sequencing based genotyping of 11 human leukocyte antigen loci
Ji Yeon Kim1, Hae-In Song1, Mi Mi Jang1, Kyoungyong Jung1, Soo Ho Park1, Kwang Joong Kim1.
1R&D Center, NGeneBio, Seoul, Korea
Introduction: Human leukocyte antigen (HLA) plays a major role in the ability of the immune system to recognize invasive, foreign and infected cells. Recently, HLA allele typing for diagnostic and therapeutic purposes has been increasing in malignant hematologic cancer and refractory autoimmune diseases. In particular, high-resolution HLA genotyping of more than 4 digits is required for bone marrow and hematopoietic stem cell transplantation in blood tumors. Here, we introduce HLAaccuTest that a novel HLA typing assay for 11 HLA loci that can be applied on illumina NGS platform to provide ultra-high resolution with unambiguous.
Materials and Methods: HLAaccuTest is based on the long-range PCR method which covers the full-length of HLA class I genes (A, B, C) and 4 loci of HLA class II genes (DQA1, DQB1, DPA1, DPB1) from promotor to 3’UTR. The 4 DRB genes (DRB1, 3, 4 and 5) covered exon 2, 3 and 4 regions. HLA typing was assigned using user friendly analysis software (EasyHLAanalyzer) that allows for automatic genotyping to four-filed resolution. EasyHLAanalyzer has been developed by our unique techniques including bioinformatic pipeline.
Results and Discussion: HLAaccuTest was validated on commercially available DNA reference panels and clinical samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in all HLA locus when compared with previously used NGS or Sanger sequencing method and the identification of several new alleles. A turnaround time of three days in average was achieved for typing 48 samples.
Conclusion: In summary, HLAaccuTest was high-resolution typing assay with high sensitivity and accuracy by NGS and can be achieved in an acceptable turnaround time for a clinical laboratory.
This work was supported by the Technology Development Program(S2642276) funded by the Ministry of SMEs and Startups(MSS, Korea)..
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