Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.

BACKGROUND: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. RESULTS: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. CONCLUSIONS: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

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.