Identification and sequencing of novel alleles: Utility of next-generation sequencing methods

Abstract

Aim Sanger sequencing-based typing (SBT) requires multiple steps to sequence novel HLA alleles, as allele-specific (AS) primers are needed to separate novel from other encoded alleles. Next-generation sequencing (NGS) includes phase information, and has the potential to sequence novel alleles without AS primers. Here we compare the process of novel allele identification and sequencing using SBT and NGS assays. Methods Novel alleles were identified in peripheral blood during routine clinical testing using Atria SBT kits (Abbot Molecular). Germline encoding was verified by typing buccal samples. For Sanger sequencing of novel alleles, AS amplification primers were designed to take advantage of nucleotide polymorphisms between encoded alleles, and patients with homozygous loci were used to verify primer specificity. Amplified products were Sanger sequenced using custom or commercially-available primers. For NGS sequencing, the Holotype X4 (Omixon) kit was used in its default configuration. Results Four novel, germline-encoded HLA alleles were identified in three patients. Novel HLA-A and B alleles were sequenced using AS amplification primers and Sanger sequencing. A novel DQB1 allele previously identified but not resolved by SBT was sequenced using the NGS assay. A novel DQB1 allele not identified by SBT, as the polymorphism was outside the covered region, was identified and sequenced by NGS. The names HLA-A∗66:22, B∗07:238, DQB1∗03:180, and DQB1∗06:01:15 have been officially assigned by the WHO Nomenclature Committee. Conclusions While SBT assays used in the clinical setting are able to identify novel alleles, subsequent sequencing is a cumbersome and time-consuming process. Furthermore, SBT assays may miss novel polymorphisms outside of the exons covered by the assay. NGS systems, on the other hand, can identify and sequence novel alleles over a larger genomic region as part of the normal clinical workflow. However, we experienced issues with the Omixon NGS kit, including the inability to sequence buccal cell DNA, and difficulties amplifying the DQB1 locus, which could have impacted clinical workflow had NGS been the primary method of testing. As the emerging technology of NGS is introduced to complement established SBT assays, it is important to evaluate the strengths and weaknesses of each.