Authors:
D. Baier (Tuebingen, DE)
H. Fischer (Tübingen, DE)
J. Wuchter (Tuebingen, DE)
A. Heidl (Dresden, DE)
I. Böhme (Dresden, DE)
V. Lange (Dresden, DE)
J. Sauter (Tuebingen, DE)
J. Hofmann (Tuebingen, DE)
A. Schmidt (Tuebingen, DE)
Since 2013, typing of DKMS donors at recruitment level has been performed by an in-house developed amplicon based NGS approach. Since this release, over 1.8 million potential unrelated stem cell donors have been typed at recruitment for HLA-A,-B,-C,-DRB1,-DQB1 and -DPB1 with over 98% high-resolution results by DKMS Germany. For these donors, more than 95,000 HLA loci have been re-typed in the context of over 17,000 CT requests. In total we found 129 discrepant typing reports of which 115 (89%) were due to genotyping errors during confirmatory typing. Only for 11% (n=14) the typing at recruitment turned out to be erroneous. 11 of these cases referred to HLA-DPB1, 2 cases to HLA-DQB1 and one to HLA-B. Issues with homozygosity handling were responsible for most DPB1 discrepancies. We observed this effect in a preliminary analysis in 2015 and optimized our software neXtype to cope with homozygosity by adjusting the detection parameters for hetero- and homozygosity. Since this optimization, no additional errors due to false detection of homozygosity have been observed. Out of 115 errors in CT, 98 (85%) comprised HLA class II loci. About half (60) of these 115 discrepancies were outside the transplantation relevant antigen recognition domain and might be due to inadequate usage of multi-allele codes for reporting typing results. 34 of the 39 DRB1 errors were related to DRB1*14:01 vs. DRB1*14:54 that could be distinguished by typing exon 3 to recognize the relevant SNP. If exon 3 is not typed, the result should be reported as DRB1*14:01:01G (or DRB1*14:BCAD). One error was related to ambiguities remaining in Sanger sequencing which are resolved by next generation sequencing. In conclusion, we were able to show that using an NGS based typing strategy is of exceptional high quality. We observed an error rate below 0.15 ‰ (14 out of 95,876) with the maximum error rate per locus not exceeding 1.6 ‰ for DPB1 (11 out of 6,921).