Full-Length Characterization of Novel HLA-DRB1 Alleles for Reference Database Submission.

The prerequisite for successful HLA genotyping is the integrity of the large allele reference database IPD-IMGT/HLA. Consequently, it is in the laboratories' best interest that the data quality of submitted novel sequences is high. However, due to its long and variable length, the gene HLA-DRB1 presents the biggest challenge and as of today only 16% of the HLA-DRB1 alleles in the database are characterized in full length. To improve this situation, we developed a protocol for long-range PCR amplification of targeted HLA-DRB1 alleles. By subsequently combining both long-read and short-read sequencing technologies, our protocol ensures phased and error-corrected sequences of reference grade quality. This dual redundant reference sequencing (DR2S) approach is of particular importance for correctly resolving the challenging repeat regions of DRB1 intron 1. Until today, we used this protocol to characterize and submit 384 full-length HLA-DRB1 sequences to IPD-IMGT/HLA.

HLA-E diversity unfolded: Identification and characterization of 170 novel HLA-E alleles.

HLA-E is a member of the nonclassical HLA class Ib genes. Even though it is structurally highly similar to the classical HLA class Ia genes, it is less diverse and only 45 alleles and 12 proteins were known in December 2019 (IPD-IMGT/HLA, release 3.38.0). Since 2017, we have genotyped over 3 million voluntary stem cell donors for HLA-E by sequencing the most relevant allele-determining bases of exons 2 and 3. As expected, most donors harbor the two predominant alleles HLA-E*01:01 and/or HLA-E*01:03. However, in 1666 (0.05%) of our samples we detected 345 distinct novel HLA-E sequences. The most frequent one was identified in 162 samples and has by now been named HLA-E*01:114. To characterize these novel alleles in full-length, we used both short-read Illumina and long-read PacBio sequencing to obtain fully phased and highly accurate sequences. This resulted in 234 submissions to IPD-IMGT/HLA comprising 170 novel HLA-E alleles, which encode for 93 novel HLA-E proteins, as well as 64 confirmations or sequence extensions. Consequently, the number of HLA-E alleles in the database (release 3.42.0) has now increased to 256 HLA-E alleles and 110 HLA-E proteins.