Abstract
Aim An HLA class II next generation sequencing (NGS) method, on the Illumina MiSeq platform with software we developed to extract HLA typing from the raw data, was applied to a cohort of hematopoietic stem cell transplant donors and recipients. Methods NGS data for DRB1/3/4/5, DQA1 and DQB1 included exons 2 and 3 and DPA1, DPB1 data was from exon 2. Legacy DRB1 and DQB1 typing of the transplant cohort was provided by the Seattle Cancer Care Alliance Clinical Immunogenetics Laboratory (CIL) and the Fred Hutchinson Cancer Research Center Clinical Research Database according to IRB-approved research protocols. The legacy HLA data was generated by various serology, SSP, SSOP and SBT methods. Discrepancy resolution involved review of CIL records and the NGS raw data and adjustments in the NGS analysis software. Results DRB1 NGS genotyping was available for 2436 specimens, and identified 53 alleles, including 10 of DRB1*04 alleles, 7 of *14, 6 each of *08, *13 and *15, 5 of *11, 3 each of *01 and *03, 2 of *12 and *16, and *07:01, *09:01 and *10:01. DQB1 NGS typing, available for 2444 specimens, identified 26 alleles, including 9 of DQB1*06, 6 of *03, 5 of *05, and 3 each of *02 and *04. These included a new DRB1 allele and 5 novel DQB1 alleles. Comparison of legacy and NGS data revealed 38 (0.78%) discordant DRB1 allele assignments, including 9 legacy data transcription errors, 8 legacy discordant alleles, and 21 of false homozygosity in the NGS results. At DQB1 the 283 (5.8%) discordant alleles included 4 legacy transcription errors, 24 legacy miscalls with exon 2 disparity and 208 legacy disparities involving exon 3 (not tested in legacy). NGS results exhibited 7 allele miscalls, 16 “script” errors involving exon 3 polymorphism, and 4 “rule set” errors in failure to detect DQB1*05:04. Conclusions The less than 1% NGS miscalls at DRB1 (0.43%) and DQB1 (0.55%) and the detection of novel allele sequences demonstrates the robust reliability and sensitivity of this next generation sequencing technology. Smith: Scisco Genetics: Consultant. Pereira: Scisco Genetics: Science Med Advisor. Geraghty: Scisco Genetics: Science Med Advisor.
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