Abstract
Aim Validation of HLA typing by next-generation sequencing (NGS) demands evidence of the assay’s accuracy compared to Sanger sequencing. We evaluated the AllType™ NGS (One Lambda) and TypeStreamVisual™NGS analysis software (One Lambda) for accuracy on HLA genotyping. Methods 48 Brazilian DNA samples selected for allelic diversity were sequenced for HLA class I and class II genes following AllType™ NGS protocol using automation of the Ion Chef and S5 sequencing system (One Lambda). Sequencing data was analyzed by TypeStreamVisual™ software 1.0.0 with IMGTHLA database 3.27.0.8. All samples were previously typed for HLA-A, B, C, DRB1, DQB1 and DPB1 by SeCore Sequence-Based Typing kits (One Lambda) using uTYPE HLA sequence analysis software (One Lambda).Evaluation was done comparing NGS results with previously assigned by Sanger’s method. Results AllTypeTM NGS and SeCore results were 100% concordant for HLA- A, B, C, DRB1, DQB1 and DPB1 loci. Following ambiguities were detected for class I and class II:B*15:01:01 B*15:03:01/B*15:39:01 B*15:54; B*07:02:01 B*08:01:01/B*08:156 B*42:01:01; DRB1*12:01:01 DRB1*13:02:01/DRB1*12:10 DRB1*13:02:01; DQB1*06:03:01 DQB1*06:09:01/DQB1*06:41 DQB1*06:88; DPB1*04:01:01 DPB1*04:02:01/DPB1*105:01 DPB1*126:01 Allelic imbalance cases occurred most in DQB1 locus, all involving DQB1*02. All null alleles required identification by the National Marrow Donor Program could be ruled out as potential results. Reproducibility was validated by 3 samples tested 3 times in the same assay run and 4 samples tested in 3 different runs. 20 blind samples sequenced also by NGS were tested and approved up to 3-field result for all loci. Conclusions We concluded that AllType™ NGS is reliable to be introduced in clinical routine for HLA-A, B, C, DRB1, DQB1 and DPB1 loci. Although HLA-B ambiguities needed additional tests, there was enormous reduction in the number of ambiguities detected by AllType™ providing highest cost benefit than Sanger sequencing. Download : Download high-res image (548KB) Download : Download full-size image
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