BackgroundA 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.ResultsWe 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.ConclusionsThe 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.

The high degree of polymorphism at HLA class I and class II loci makes high resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results due to incomplete genomic coverage and inability to set phase for HLA haplotype determination. The 454 Life Sciences GS FLX next generation sequencing system coupled with Conexio ATF software can provide very high resolution HLA genotyping. High throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, and DRB3, DRB4 and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs (bp) and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and Conexio ATF software allows reliable identification of HLA genotypes at high resolution.

Homozygous carriers of CCR5-Δ32, a gene variant of CC-type chemokine receptor 5 (CCR5), are highly resistant to infections with human immunodeficiency virus type 1 (HIV-1) and therefore preferred stem cell donors for HIV-infected patients. We analyzed CCR5 typing data of 1,333,035 potential hematopoietic stem cell donors enlisted with three national DKMS donor centers. Allele and genotype frequencies were determined for 87 countries of origin as self-assessed by the donors. CCR5-Δ32 allele frequencies ranged from 16.4% in the Norwegian sample to 0 in donors from Ethiopia. The highest CCR5-Δ32/Δ32 genotype frequency was found in the sample from the Faroe Islands (2.3%), whereas in 27 samples, predominantly of donors from Africa, Asia and South America, none of the individuals carried this genotype. The characteristic CCR5-Δ32 allele frequency decline from Northern to Southeastern Eurasia supports findings of earlier studies. With available HLA haplotype frequency information for the patient's ethnicity, our data allows upfront estimation of the probability that an HLA-matched donor with CCR5-Δ32/Δ32 genotype can be found for a patient in need of hematopoietic stem cell transplantation.

BackgroundAt the DKMS Life Science Lab, Next Generation Sequencing (NGS) has been used for ultra-high-volume high-resolution genotyping of HLA loci for the last three and a half years. Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments.ResultsBetween January 2013 and June 2016, 2,714,110 samples largely from German, Polish and UK-based potential stem cell donors have been processed. 98.9% of all alleles for the targeted HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) were typed at high resolution or better. Initially a simple three-step workflow based on nanofluidic chips in conjunction with 4-primer amplicon tagging was used. Over time, we found that this setup results in PCR artefacts such as primer dimers and PCR-mediated recombination, which may necessitate repeat typing. Split workflows for low- and high-DNA-concentration samples helped alleviate these problems and reduced average per-locus repeat rates from 3.1 to 1.3%. Further optimisations of the workflow included the use of phosphorothioate oligos to reduce primer degradation and primer dimer formation, and employing statistical models to predict read yield from initial template DNA concentration to avoid intermediate quantification of PCR products. Finally, despite the populations typed at DKMS Life Science Lab being relatively homogenous genetically, an analysis of 1.4 million donors processed between January 2015 and May 2016 led to the discovery of 1,919 distinct novel HLA alleles.ConclusionsAmplicon-based NGS HLA genotyping workflows have become the workhorse in high-volume tissue typing of registry donors. The optimisation of workflow practices over multiple years has led to insights and solutions that improve the efficiency and robustness of short amplicon based genotyping workflows.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3575-z) contains supplementary material, which is available to authorized users.

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