P126 Towards full length sequencing

Abstract

Aim Excluding null alleles and resolving ambiguities within G groups are mandatory in the laboratories serving unrelated HSCT programs. Since routine sequencing strategies have mainly focused on exons 2 and 3, for more than 90% of the HLA alleles valuable full-length sequence information is lacking. The 17th IHIWG programme includes working groups for full length HLA gene sequencing. We would like to contribute to the component with obtaining full-length sequences of rare alleles encountered in our laboratory. Methods Using the full-length hemizygous Sanger sequencing method (SSBT 17th workshop protocol) we extended the sequence of a new allele, DNA carrying allele, B∗27:30 found in the family of Slovenian origin and of 6 samples of individuals with family confirmed haplotype A∗02:01-B∗27:05-DRB1∗01:01, common in Slovenian population. Moreover, Oxford Nanopore MinION® device, a small and low-cost single-molecule sequencer, which offers the possibility of sequencing long DNA fragments, was used for retyping all samples as well as for additional nine randomly chosen individuals previously typed by Sanger SBT (AlleleSEQR HLA-SBT Reagents, Abbott) and another NGS platform (Roche reagents with the Roche GS Junior system). Results We obtained complete HLA-A, B and C full length sequences of all analysed samples with SSBT that provide reference data for NGS. Raw data produced by MinION were concordant with SSBT as well as with EFI standards requirements enabling allelic resolution typing result interpreted by adequate commercial software. Conclusions In conclusion, we find SBT with Oxford Nanopore MinION® device as very promising method to be used both in high throughput, but definitely also in medium to small size H&I laboratories.