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 to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48 amplicons were sequenced full length in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was performed using the SMRT portal. Sequence analysis was performed using the NGSengine software (GenDx). Results: We analyzed a set of 48 randomly picked samples. With 3 exceptions due to PCR failure, all genotype assignments conformed to standard genotyping results based on exons 2 and 3. Allelic proportions for heterozygous positions were evenly distributed (range 0.4 – 0.6) for all samples, suggesting unbiased amplifications. Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. We describe novel intronic sequence variation of the 7 so far genomically defined alleles, as well as 7 whole-length DPB1 alleles with hitherto unknown intronic regions. One of these alleles (HLA-DPB1*131:01) is classified as rare. Conclusion: Here we present a whole gene amplification and sequencing workflow for DPB1 alleles utilizing single molecule real-time (SMRT) sequencing from Pacific Biosciences. Validation of consensus sequences against known exonic sequences highlights the reliability of this technology. This workflow will facilitate amending the IMGT/HLA Database for DPB1.
Organization: DKMS Life Science Lab