Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can…
Aim: In contrast to exon-based HLA-typing approaches, whole gene genotyping crucially depends on full-length sequences submitted to the IMGT/HLA Database. Currently, full-length sequences are provided for only 7 out of 520 HLA-DPB1 alleles. Therefore, we developed a fully phased whole-gene sequencing approach for DPB1, to facilitate further exploration of the allelic structure at this locus. Methods: Primers were developed flanking the UTR-regions of DPB1 resulting in a 12 kb amplicon. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene-specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated…
Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated “high resolution” (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2-4.4 kb. Using a…